Method of detecting an analyte in a sample using semiconductor nanocrystals as detectable label

ABSTRACT

The use of semiconductor nanocrystals as detectable labels in various chemical and biological applications is disclosed. The methods find use for detecting a single analyte, as well as multiple analytes by using more than one semiconductor nanocrystal as a detectable label, each of which emits at a distinct wavelength.

CROSS-REFERENCE TO RELATED APPLICATION

This application is related to provisional patent application Ser. No.60/133,084, filed May 7, 1999, from which priority is claimed under 35USC §119(e)(1) and which is incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

The present invention relates generally to the detection of analytes ina sample. In particular, the invention relates to assays that usesemiconductor nanocrystals as a detectable label. The invention furtherrelates to assays in which multiple analytes can be detectedsimultaneously by using more than one semiconductor nanocrystal as adetectable label, each of which emits at a distinct wavelength.

BACKGROUND OF THE INVENTION

A variety of chemical and biological assays exist to identify an analyteof interest in a given sample. For example, immunoassays, such asenzyme-linked immunosorbent assays (ELISAs) are used in numerousdiagnostic, research and screening applications. In its most commonform, an ELISA detects the presence and/or concentration of an analytein a sample using an antibody which specifically recognizes the analyte.An enzyme label, capable of providing a detectable signal, is conjugatedto the antibody. The analyte is either immobilized directly onto a solidsupport (direct-capture ELISA) or is bound to a different specificantibody which itself is immobilized on a solid support. The presence ofthe immobilized analyte is detected by binding to it the detectablylabeled antibody. A variety of different ELISA formats have beendescribed. See, e.g., U.S. Pat. Nos. 4,011,308 to Giaever, 4,722,890 toSanders et al., Re. 032696 to Schuurs et al., 4,016,043 to Schuurs etal., 3,876,504 to Koffler, 3,770,380 to Smith, and 4,372,745 to Mandleet al.

Another technique for detecting biological compounds is fluorescence insitu hybridization (FISH). Swiger et al. (1996) Environ. Mol. Mutagen.27:245-254; Raap (1998) Mut. Res. 400:287-298; Nath et al. (1997)Biotechnic. Histol. 73:6-22. FISH allows detection of a predeterminedtarget oligonucleotide, e.g., DNA or RNA, within a cellular or tissuepreparation by, for example, microscopic visualization. Thus, FISH is animportant tool in the fields of, for example, molecular cytogenetics,pathology and immunology in both clinical and research laboratories.

This method involves the fluorescent tagging of an oligonucleotide probeto detect a specific complementary DNA or RNA sequence. Specifically,FISH involves incubating an oligonucleotide probe comprising anoligonucleotide that is complementary to at least a portion of thetarget oligonucleotide with a cellular or tissue preparation containingor suspected of containing the target oligonucleotide. A detectablelabel, e.g., a fluorescent dye molecule, is bound to the oligonucleotideprobe. A fluorescence signal generated at the site of hybridization istypically visualized using an epi fluorescence microscope. Analternative approach is to use an oligonucleotide probe conjugated withan antigen such as biotin or digoxygenin and a fluorescently taggedantibody directed toward that antigen to visualize the hybridization ofthe probe to its DNA target. A variety of FISH formats are known in theart. See, e.g., Dewald et al. (1993) Bone Marrow Transplantation12:149-154; Ward et al. (1993) Am. J. Hum. Genet. 52:854-865; Jalal etal. (1998) Mayo Clin. Proc. 73:132-137; Zahed et al. (1992) Prenat.Diagn. 12:483-493; Kitadai et al. (1995) Clin. Cancer Res. 1:1095-1102;Neuhaus et al. (1999) Human Pathol. 30:81-86; Hack et al., eds., (1980)Association of Cytogenetic Technologists Cytogenetics Laboratory Manual.(Association of Cytogenetic Technologists, San Francisco, Calif.); Bunoet al. (1998) Blood 92:2315-2321; Patterson et al. (1993) Science260:976-979; Patterson et al. (1998) Cytometry 31:265-274; Borzi et al.(1996) J. Immunol. Meth. 193:167-176; Wachtel et al. (1998) Prenat.Diagn. 18:455-463; Bianchi (1998) J. Perinat. Med. 26:175-185; and Munne(1998) Mol. Hum. Reprod. 4:863-870.

FISH provides a powerful tool for the chromosomal localization of geneswhose sequences are partially or fully known. Other applications of FISHinclude in situ localization of mRNA in tissues sample and localizationof nongenetic DNA sequences such as telomeres.

Signal amplification is yet another method for sensitive detection ofnucleic acids and other receptor/ligand interactions. Direct detectionof a target nucleic acid is possible by hybridization of a complementarynucleic acid probe to the target. Detection of the complex can beachieved by numerous means, e.g., a labeled probe or a reagent dye thatspecifically attaches to the target/probe complex. Such “direct”detection systems are often not sensitive enough to detect a targetnucleic acid in a biological sample. One method for overcoming thislimitation is to employ signal amplification. Signal amplification canbe done, for example, by indirectly binding multiple signal-generatingmolecules to an analyte through a molecule which is (1) complementary tothe analyte and (2) contains multiple signal-generating molecule bindingsites and which signal-generating molecules (i) contain a detectablelabel, (ii) bind to or otherwise activate a label or (iii) contain sitesfor binding additional layers of molecules which may in turn facilitategeneration of a detectable signal. Thus, rather than a singlesignal-generating label associated with the target molecule, signalamplification results in the association of multiple signal-generatinglabels associated with the target molecule and, therefore, enhancedassay sensitivity.

Nucleic acid hybridization assays are described in, for example, U.S.Pat. Nos. 5,681,697 to Urdea et al., 5,124,246 to Urdea et al.,4,868,105 to Urdea et al., and European Patent Publication No. 70.685,inventors Heller et al.

There are many assays designed to obtain the sequence of a DNA sample.Each of these methods shares some or all of a set of common features.These features include: sequence specificity derived from complementaryoligonucleotide hybridization or annealing; a solid support or solidphase which allows separation of specifically bound assay reagents; anda label which is used for detecting the presence or absence of thespecific, intended assay interaction. Examples of assays designed todetect the sequence of a DNA sample can be found in U.S. Pat. Nos.5,888,731 to Yager et al., 5,830,711 to Barany et al., 5,800,994 toMartinelli et al., 5,792,607 to Backman et al., 5,716,784 to Di Cesare,5,578,458 to Caskey et al., 5,494,810 to Barany et al., 4,925,785 toWang et al., 4,9898,617 to Landegren et al.

Chemical compounds are typically evaluated for potential therapeuticutility by assaying their ability to affect, for example, enzymeactivity, ligand-receptor interactions, protein-protein interactions, orthe like. Evaluating the effect of each individual candidate compound ona variety of systems can be tedious and time-consuming. Accordingly,protocols have been developed to evaluate rapidly multiple candidatecompounds in a particular system and/or a candidate compound in aplurality of systems. Such protocols for evaluating candidate compoundshave been referred to as high throughput screening (HTS).

In one typical protocol, HTS involves the dispersal of a candidatecompound into a well of a multiwell cluster plate, for example, a96-well or higher format plate, e.g., a 384-, 864-, or 1536-well plate.The effect of the compound is evaluated on the system in which it isbeing tested. The “throughput” of this technique, i.e., the combinationof the number of candidate compounds that can be screened and the numberof systems against which candidate compounds can be screened, is limitedby a number of factors, including, but not limited to: only one assaycan be performed per well; if conventional dye molecules are used tomonitor the effect of the candidate compound, multiple excitationsources are required if multiple dye molecules are used; and as the wellsize becomes small (e.g., the 1536-well plate can accept about 5 μl oftotal assay volume), consistent dispensing of individual components intoa well is difficult and the amount of signal generated by each assay issignificantly decreased, scaling with the volume of the assay.

A number of assay formats can be used for HTS assays. For example, theinhibitory effect of the candidate compound on, e.g., enzyme activity,ligand-receptor binding, and the like, can be measured by comparing theendpoint of the assay in the presence of a known concentration of thecandidate to a reference which is performed in the absence of thecandidate and/or in the presence of a known inhibitor compound. Thus,for example, a candidate compound can be identified which inhibits thebinding of a ligand and its receptor, or which inhibits enzyme activity,decreasing the turnover of the enzymatic process. When this process (theinhibited process) is of clinical significance, the candidates areidentified to be potential drugs for a particular condition.

A 1536-well plate is merely the physical segregation of sixteen assayswithin a single 96 well plate format. It would be advantageous tomultiplex 16 assays into a single well of the 96 well plate. This wouldresult in greater ease of dispensing reagents into the wells and in highsignal output per well. In addition, performing multiple assays in asingle well allows simultaneous determination of the potential of acandidate compound to affect a plurality of target systems. Using HTSstrategies, a single candidate compound can be screened for activity as,e.g., a protease inhibitor, an inflammation inhibitor, an antiasthmatic,and the like, in a single assay.

Each of the above-described assay formats utilizes detectable labels toidentify the analyte of interest. Radiolabeled molecules and compoundsare frequently used to detect biological compounds both in vivo and invitro. However, due to the inherent problems associated with the use ofradioactive isotopes, nonradioactive methods of detecting biological andchemical compounds are often preferable.

For example, fluorescent molecules are commonly used as tags fordetecting an analyte of interest. Fluorescence is the emission of lightresulting from the absorption of radiation at one wavelength(excitation) followed by nearly immediate reradiation usually at adifferent wavelength (emission). Organic fluorescent dyes are typicallyused in this context. However, there are chemical and physicallimitations to the use of such dyes. One of these limitations is thevariation of excitation wavelengths of different colored dyes. As aresult, the simultaneous use of two or more fluorescent tags withdifferent excitation wavelengths requires multiple excitation lightsources.

Another drawback of organic dyes is the deterioration of fluorescenceintensity upon prolonged and/or repeated exposure to excitation light.This fading, called photobleaching, is dependent on the intensity of theexcitation light and the duration of the illumination. In addition,conversion of the dye into a nonfluorescent species is irreversible.Furthermore, the degradation products of dyes are organic compoundswhich may interfere with the biological processes being examined.

Additionally, spectral overlap exists from one dye to another. This isdue, in part, to the relatively wide emission spectra of organic dyesand the overlap of the spectra near the tailing region. Few lowmolecular weight dyes have a combination of a large Stokes shift, whichis defined as the separation of the absorption and emission maxima, andhigh fluorescence output. In addition, low molecular weight dyes may beimpractical for some applications because they do not provide a brightenough fluorescent signal.

Furthermore, the differences in the chemical properties of standardorganic fluorescent dyes make multiple, parallel assays impractical asdifferent chemical reactions may be involved for each dye used in thevariety of applications of fluorescent labels.

Thus, there is a continuing need in the assay art for labels with thefollowing features: (i) high fluorescent intensity (for detection insmall quantities), (ii) adequate separation between the absorption andemission frequencies, (iii) good solubility, (iv) ability to be readilylinked to other molecules, (v) stability towards harsh conditions andhigh temperatures, (vi) a symmetric, nearly gaussian emission lineshapefor easy deconvolution of multiple colors, and (vii) compatibility withautomated analysis. At present, none of the conventional fluorescentlabels satisfies all of these requirements.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that semiconductornanocrystals can be used as reliable and sensitive detectable labels ina variety of biological and chemical formats. Semiconductor nanocrystals(also know as quantum dot and Qdot™ nanocrystals) can be produced thathave characteristic spectral emissions. These spectral emissions can betuned to a desired energy by varying the particle size, sizedistribution and/or composition of the particle. A targeting compoundthat has affinity for one or more selected biological or chemicaltargets is associated with the semiconductor nanocrystal. Thus, thesemiconductor nanocrystal will interact or associate with the target dueto the affinity of the targeting compound for the target. The locationand/or nature of the association can be determined, for example, byirradiation of the sample with an energy source, such as an excitationlight source. The semiconductor nanocrystal emits a characteristicemission spectrum which can be observed and measured, for example,spectroscopically.

Conveniently, emission spectra of a population of semiconductornanocrystals can be manipulated to have linewidths as narrow as 25-30nm, depending on the size distribution heterogeneity of the samplepopulation, and lineshapes that are symmetric, gaussian or nearlygaussian with an absence of a tailing region. Accordingly, the abovetechnology allows for detection of one, or even several, differentbiological or chemical moieties in a single reaction. The combination oftunability, narrow linewidths, and symmetric emission spectra without atailing region provides for high resolution of multiply sizednanocrystals, e.g., populations of monodisperse semiconductornanocrystals having multiple distinct size distributions within asystem, and simultaneous detection of a variety of biological moieties.

In addition, the range of excitation wavelengths of such nanocrystals isbroad and can be higher in energy than the emission wavelengths of allavailable semiconductor nanocrystals. Consequently, this allows the useof a single energy source, such as light, usually in the ultraviolet orblue region of the spectrum, to effect simultaneous excitation of allpopulations of semiconductor nanocrystals in a system having distinctemission spectra. Semiconductor nanocrystals are also more robust thanconventional organic fluorescent dyes and are more resistant tophotobleaching than the organic dyes. The robustness of the nanocrystalalso alleviates the problem of contamination of degradation products ofthe organic dyes in the system being examined. Therefore, the presentinvention provides uniquely valuable tags for detection of biologicaland chemical molecules.

Accordingly, in one embodiment, the invention is directed to a method ofdetecting one or more target analytes in a sample containing orsuspected of containing the one or more analytes, comprising the stepsof:

(a) providing the sample on a solid support;

(b) combining the sample with a semiconductor nanocrystal conjugate,wherein the combining is performed under conditions that allow formationof a complex comprising the conjugate and the analyte, when present;

(c) removing any unbound conjugate; and

(d) detecting the presence of the complex, if present, by monitoring aspectral emission mediated by the semiconductor nanocrystal in thecomplex, wherein the emission indicates the presence of one or moretarget analytes in the sample.

In certain embodiments, the invention is directed to a method wherethere is a plurality of target analytes and the method furthercomprises:

(a) providing a conjugate specific for each target analyte, wherein eachsemiconductor nanocrystal conjugate has an emission spectrum distinctfrom the other semiconductor nanocrystal conjugates; and

(b) detecting the presence of the target analytes by monitoring thespectral emissions of the sample, wherein the emissions indicate thepresence of the target analytes in the sample.

In yet another embodiment, the invention is directed to a method ofdetecting one or more target analytes in a sample containing orsuspected of containing the one or more analytes, comprising the stepsof:

(a) providing an unlabeled specific-binding molecule on a solid support;

(b) combining the sample with the specific-binding molecule, wherein thecombining is performed under conditions that allow formation of a firstcomplex comprising the specific-binding molecule and the analyte, whenpresent;

(c) removing any unbound sample;

(d) combining the first complex with a semiconductor nanocrystalconjugate, wherein the combining is performed under conditions thatallow formation of a second complex comprising the conjugate and theanalyte, when present;

(e) removing any unbound conjugate;

(f) detecting the presence of the second complex, if present, bymonitoring a spectral emission mediated by the semiconductor nanocrystalin the second complex, wherein the emission indicates the presence ofone or more target analytes in the sample.

In certain embodiments, the method is one where there is a plurality oftarget analytes and the method further comprises:

(a) providing an unlabeled specific-binding molecule on a solid supportspecific for each target analyte and providing a semiconductornanocrystal conjugate specific for each target analyte, wherein eachsemiconductor nanocrystal conjugate has an emission spectrum distinctfrom the other semiconductor nanocrystal conjugates; and

(b) detecting the presence of the target analytes by monitoring thespectral emissions of the sample, wherein the emissions indicate thepresence of the target analytes in the sample.

In yet another embodiment, the invention is directed to a method ofdetecting one or more target analytes in a sample containing orsuspected of containing the one or more analytes, comprising the stepsof:

(a) providing the sample on a solid support;

(b) combining with the sample a specific-binding molecule, wherein (i)the specific-binding molecule comprises a first member of a bindingpair, and (ii) the combining is performed under conditions that allowformation of a first complex comprising the specific-binding moleculeand the analyte, when present;

(c) removing any unbound specific-binding molecule;

(d) combining the first complex with a second member of the bindingpair, wherein (i) the second member of the binding pair is linked to afirst semiconductor nanocrystal; and (ii) the combining is performedunder conditions that allow formation of a second complex comprising thebinding pair and the one or more analytes;

(e) detecting the presence of the second complex, if present, bymonitoring a spectral emission mediated by the first semiconductornanocrystal in the second complex, wherein the emission indicates thepresence of one or more target analytes in the sample.

In certain embodiments, the first member of the binding pair is a firstantibody and the second member of the binding pair is a second antibodyreactive with the first antibody; or the first member of the bindingpair is biotin and the second member of the binding pair isstreptavidin; or the first member of the binding pair is digoxygenin andthe second member of the binding pair is an antibody directed againstdigoxygenin; or the first member of the binding pair is flourescein andthe second member of the binding pair is an antibody directed againstflourescein.

In another embodiment, the above method is one where there is more thanone analyte, and the method further comprises:

combining with the sample a second specific-binding molecule, wherein(i) the second specific-binding molecule comprises a first member of asecond binding pair, and (ii) the combining is performed underconditions that allow formation of a third complex comprising the secondspecific-binding molecule and the analyte, when present;

removing any unbound second specific-binding molecule;

combining the third complex with a second member of the second bindingpair, wherein (i) the second member of the second binding pair is linkedto a second semiconductor nanocrystal that has an emission spectrumdistinct from the first semiconductor nanocrystal; and (ii) thecombining is performed under conditions that allow formation of a fourthcomplex comprising the second binding pair and an analyte; and

detecting the presence of the fourth complex, if present, by monitoringa second spectral emission mediated by the second semiconductornanocrystal in the fourth complex, wherein the second emission indicatesthe presence of more than one target analyte in the sample.

In still a further embodiment, the invention is directed to a method ofdetecting one or more target analytes in a sample containing orsuspected of containing the one or more analytes. The method comprisesthe steps of:

(a) providing a first complex comprising at least one specific-bindingmolecule to which is bound a semiconductor nanocrystal conjugate,wherein the semiconductor nanocrystal has a characteristic spectralemission and wherein the conjugate specifically binds to thespecific-binding molecule;

(b) combining the sample with the first complex, wherein the combiningis performed under conditions that allow formation of a second complexcomprising the specific-binding molecule and the analyte, when present;

(c) detecting the presence of the second complex, if present, bymonitoring the characteristic spectral emission of the semiconductornanocrystal, wherein a change in the characteristic spectral emissionindicates the presence of one or more target analytes in the sample.

In certain embodiments of the method above, the plurality of firstcomplexes are provided each comprising a different specific-bindingmolecule each bound to a conjugate which specifically binds eachspecific-binding molecule and where each specific-binding molecule bindsa different analyte, and wherein each conjugate bound to a differentspecific-binding molecule comprises a semiconductor nanocrystal that hasa characteristic spectral emission distinct from the other semiconductornanocrystals; and wherein changes in the spectral emission of anyselected semiconductor nanocrystal associated with a particularspecific-binding molecule in a first complex, indicates the presence ofan analyte that binds to the particular specific-binding molecule.

In certain embodiments, the specific-binding molecule is radiolabeled,and when the conjugate is bound to the specific-binding molecule thesemiconductor nanocrystal emits light, as described in Example 13.Further, the method may be one wherein a plurality of first complexesare provided each comprising a different specific-binding molecule eachbound to a conjugate which specifically binds each specific-bindingmolecule and where each specific-binding molecule binds a differentanalyte, and wherein each conjugate bound to a differentspecific-binding molecule comprises a semiconductor nanocrystal that hasa characteristic spectral emission distinct from the other semiconductornanocrystals; and wherein changes in the spectral emission of anyselected semiconductor nanocrystal associated with a particularspecific-binding molecule in a first complex, indicates the presence ofan analyte that binds to the particular specific-binding molecule.

In the methods above, the one or more analytes may be one or morepolypeptides or nucleic acid sequences, either the same or different;the nucleic acid sequences may be present on one or more chromosomalfragments; the nucleic acid sequences may be DNA or RNA; thesemiconductor nanocrystal conjugate may comprise an antibody, anaptamer, or at least one polymerase chain reaction primer; thespecific-binding molecule may comprise a nucleic acid molecule, e.g., anoligonucleotide, DNA, RNA, an aptamer, a protein, a receptor, anantibody, a polysaccharide or a small molecule

These and other embodiments of the present invention will readily occurto those of ordinary skill in the art in view of the disclosure herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a pictorial representation of a Qdot™ immunosorbent assay(QISA). FIG. 1A is a pictorial representation of a sandwich QISA asdescribed in Example 1. FIG. 1B is a pictorial representation of adirect capture QISA as described in Example 2. FIG. 1C is a pictorialrepresentation of a fluid-phase QISA as described in Example 3.

FIG. 2 shows the results of a QISA performed on a 96-well plate and readin a standard fluorescent plate reader, as described in Example 1A.

FIG. 3 shows the distribution of beads among three rows (A, B and C) ofeight wells with differing concentrations of semiconductor nanocrystalconjugates, as described in Example 12.

FIG. 4 is a representation of results that can be obtained whensemiconductor nanocrystals are used in a homogeneous scintillationinhibition assay, as described in Example 13. In the Figure, lanes A, Band C contain different inhibitors.

FIG. 5 is a pictorial representation of a competitive microsphere filterassay as described in Example 19.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry and biochemistry within theskill of the art. Such techniques are explained fully in the literature.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural references unless the contentclearly dictates otherwise. Thus, for example, reference to “asemiconductor nanocrystal” includes a mixture of two or more suchsemiconductor nanocrystals, an “analyte” includes more than one suchanalyte, and the like.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

I. DEFINITIONS

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

The terms “semiconductor nanocrystal,” “quantum dot” and “Qdot™nanocrystal” are used interchangeably herein and refer to an inorganiccrystallite between about 1 nm and about 1000 nm in diameter or anyinteger or fraction of an integer therebetween, preferably between about2 nm and about 50 nm or any integer or fraction of an integertherebetween, more preferably about 2 nm to about 20 nm (such as about2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20nm). A semiconductor nanocrystal is capable of emitting electromagneticradiation upon excitation (i.e., the semiconductor nanocrystal isluminescent) and includes a “core” of one or more first semiconductormaterials, and may be surrounded by a “shell” of a second semiconductormaterial. A semiconductor nanocrystal core surrounded by a semiconductorshell is referred to as a “core/shell” semiconductor nanocrystal. Thesurrounding “shell” material will preferably have a bandgap energy thatis larger than the bandgap energy of the core material and may be chosento have an atomic spacing close to that of the “core” substrate. Thecore and/or the shell can be a semiconductor material including, but notlimited to, those of the group II-VI (ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe,HgS, HgSe, HgTe, MgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS,BaSe, BaTe, and the like) and III-V (GaN, GaP, GaAs, GaSb, InN, InP,InAs, InSb, and the like) and IV (Ge, Si, and the like) materials, andan alloy or a mixture thereof.

A semiconductor nanocrystal is, optionally, surrounded by a “coat” of anorganic capping agent. The organic capping agent may be any number ofmaterials, but has an affinity for the semiconductor nanocrystalsurface. In general, the capping agent can be an isolated organicmolecule, a polymer (or a monomer for a polymerization reaction), aninorganic complex, and an extended crystalline structure. The coat isused to convey solubility, e.g., the ability to disperse a coatedsemiconductor nanocrystal homogeneously into a chosen solvent,functionality, binding properties, or the like. In addition, the coatcan be used to tailor the optical properties of the semiconductornanocrystal. Methods for producing capped semiconductor nanocrystals arediscussed further below.

Thus, the terms “semiconductor nanocrystal,” “quantum dot” and “Qdot™nanocrystal” as used herein denote a coated semiconductor nanocrystalcore, as well as a core/shell semiconductor nanocrystal.

By “luminescence” is meant the process of emitting electromagneticradiation (light) from an object. Luminescence results from a systemwhich is “relaxing” from an excited state to a lower state with acorresponding release of energy in the form of a photon. These statescan be electronic, vibronic, rotational, or any combination of thethree. The transition responsible for luminescence can be stimulatedthrough the release of energy stored in the system chemically or addedto the system from an external source.

The external source of energy can be of a variety of types includingchemical, thermal, electrical, magnetic, electromagnetic, physical orany other type capable of causing a system to be excited into a statehigher than the ground state. For example, a system can be excited byabsorbing a photon of light, by being placed in an electrical field, orthrough a chemical oxidation-reduction reaction. The energy of thephotons emitted during luminescence can be in a range from low-energymicrowave radiation to high-energy x-ray radiation. Typically,luminescence refers to photons in the range from UV to IR radiation.

“Monodisperse particles” include a population of particles wherein atleast about 60% of the particles in the population, more preferably 75%to 90% of the particles in the population, or any integer in betweenthis range, fall within a specified particle size range. A population ofmonodispersed particles deviate less than 10% rms (root-mean-square) indiameter and preferably less than 5% rms.

The phrase “one or more sizes of semiconductor nanocrystals” is usedsynonymously with the phrase “one or more particle size distributions ofsemiconductor nanocrystals.” One of ordinary skill in the art willrealize that particular sizes of semiconductor nanocrystals are actuallyobtained as particle size distributions.

By use of the term “a narrow wavelength band” or “narrow spectrallinewidth” with regard to the electromagnetic radiation emission of thesemiconductor nanocrystal is meant a wavelength band of emissions notexceeding about 40 nm, and preferably not exceeding about 20 nm in widthand symmetric about the center, in contrast to the emission bandwidth ofabout 100 nm for a typical dye molecule with a red tail which may extendthe bandwidth out as much as another 100 nm. It should be noted that thebandwidths referred to are determined from measurement of the full widthof the emissions at half peak height (FWHM), and are appropriate in therange of 200 nm to 2000 nm.

By use of the term “a broad wavelength band,” with regard to theexcitation of the semiconductor nanocrystal is meant absorption ofradiation having a wavelength equal to, or shorter than, the wavelengthof the onset radiation (the onset radiation is understood to be thelongest wavelength (lowest energy) radiation capable of being absorbedby the semiconductor nanocrystal). This onset occurs near to, but atslightly higher energy than the “narrow wavelength band” of theemission. This is in contrast to the “narrow absorption band” of dyemolecules which occurs near the emission peak on the high energy side,but drops off rapidly away from that wavelength and is often negligibleat wavelengths further than 100 nm from the emission.

The terms “polynucleotide,” “oligonucleotide,” “nucleic acid” and“nucleic acid molecule” are used herein to include a polymeric form ofnucleotides of any length, either ribonucleotides ordeoxyribonucleotides. This term refers only to the primary structure ofthe molecule. Thus, the term includes triple-, double- andsingle-stranded DNA, as well as triple-, double- and single-strandedRNA. It also includes modifications, such as by methylation and/or bycapping, and unmodified forms of the polynucleotide. More particularly,the terms “polynucleotide,” “oligonucleotide,” “nucleic acid” and“nucleic acid molecule” include polydeoxyribonucleotides (containing2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), any othertype of polynucleotide which is an N- or C-glycoside of a purine orpyrimidine base, and other polymers containing normucleotidic backbones,for example, polyamide (e.g., peptide nucleic acids (PNAs)) andpolymorpholino (commercially available from the Anti-Virals, Inc.,Corvallis, Oregon, as Neugene) polymers, and other syntheticsequence-specific nucleic acid polymers providing that the polymerscontain nucleobases in a configuration which allows for base pairing andbase stacking, such as is found in DNA and RNA. There is no intendeddistinction in length between the terms “polynucleotide,”“oligonucleotide,” “nucleic acid” and “nucleic acid molecule,” and theseterms will be used interchangeably. These terms refer only to theprimary structure of the molecule. Thus, these terms include, forexample, 3′-deoxy-2′,5′-DNA, oligodeoxyribonucleotide N3′ P5′phosphoramidates, 2′-O-alkyl-substituted RNA, double- andsingle-stranded DNA, as well as double- and single-stranded RNA, DNA:RNAhybrids, and hybrids between PNAs and DNA or RNA, and also include knowntypes of modifications, for example, labels which are known in the art,methylation, “caps,” substitution of one or more of the naturallyoccurring nucleotides with an analog, internucleotide modifications suchas, for example, those with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.),with negatively charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), and with positively charged linkages (e.g.,aminoalklyphosphoramidates, aminoalkylphosphotriesters), thosecontaining pendant moieties, such as, for example, proteins (includingnucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.),those with intercalators (e.g., acridine, psoralen, etc.), thosecontaining chelators (e.g., metals, radioactive metals, boron, oxidativemetals, etc.), those containing alkylators, those with modified linkages(e.g., alpha anomeric nucleic acids, etc.), as well as unmodified formsof the polynucleotide or oligonucleotide. In particular, DNA isdeoxyribonucleic acid.

The terms “polynucleotide analyte” and “nucleic acid analyte” are usedinterchangeably and include a single- or double-stranded nucleic acidmolecule that contains a target nucleotide sequence. The analyte nucleicacids may be from a variety of sources, e.g., biological fluids orsolids, chromosomes, food stuffs, environmental materials, etc., and maybe prepared for the hybridization analysis by a variety of means, e.g.,proteinase K/SDS, chaotropic salts, or the like.

As used herein, the term “target nucleic acid region” or “targetnucleotide sequence” includes a probe-hybridizing region containedwithin the target molecule. The term “target nucleic acid sequence”includes a sequence with which a probe will form a stable hybrid underdesired conditions.

As used herein, the term “nucleic acid probe” includes reference to astructure comprised of a polynucleotide, as defined above, that containsa nucleic acid sequence complementary to a nucleic acid sequence presentin the target nucleic acid analyte. The polynucleotide regions of probesmay be composed of DNA, and/or RNA, and/or synthetic nucleotide analogs.

It will be appreciated that the hybridizing sequences need not haveperfect complementarity to provide stable hybrids. In many situations,stable hybrids will form where fewer than about 10% of the bases aremismatches, ignoring loops of four or more nucleotides. Accordingly, asused herein the term “complementary” refers to an oligonucleotide thatforms a stable duplex with its “complement” under assay conditions,generally where there is about 90% or greater homology.

The term “aptamer” (or nucleic acid antibody) is used herein to refer toa single- or double-stranded DNA or a single-stranded RNA molecule thatrecognizes and binds to a desired target molecule by virtue of itsshape. See, e.g., PCT Publication Nos. WO92/14843, WO91/19813, andWO92/05285, the disclosures of which are incorporated by referenceherein.

“Polypeptide” and “protein” are used interchangeably herein and includea molecular chain of amino acids linked through peptide bonds. The termsdo not refer to a specific length of the product. Thus, “peptides,”“oligopeptides,” and “proteins” are included within the definition ofpolypeptide. The terms include post-translational modifications of thepolypeptide, for example, glycosylations, acetylations, phosphorylationsand the like. In addition, protein fragments, analogs, mutated orvariant proteins, fusion proteins and the like are included within themeaning of polypeptide.

As used herein, the term “binding pair” refers to first and secondmolecules that specifically bind to each other. “Specific binding” ofthe first member of the binding pair to the second member of the bindingpair in a sample is evidenced by the binding of the first member to thesecond member, or vice versa, with greater affinity and specificity thanto other components in the sample. The binding between the members ofthe binding pair is typically noncovalent. Unless the context clearlyindicates otherwise, the terms “affinity molecule” and “target analyte”are used herein to refer to first and second members of a binding pair,respectively.

Exemplary binding pairs include any haptenic or antigenic compound incombination with a corresponding antibody or binding portion or fragmentthereof (e.g., digoxigenin and anti-digoxigenin; fluorescein andanti-fluorescein; dinitrophenol and anti-dinitrophenol;bromodeoxyuridine and anti-bromodeoxyuridine; mouse immunoglobulin andgoat anti-mouse immunoglobulin) and nonimmunological binding pairs(e.g., biotin-avidin, biotin-streptavidin, hormone [e.g., thyroxine andcortisol]-hormone binding protein, receptor-receptor agonist orantagonist (e.g., acetylcholine receptor-acetylcholine or an analogthereof) IgG-protein A, lectin-carbohydrate, enzyme-enzyme cofactor,enzyme-enzyme-inhibitor, and complementary polynucleotide pairs capableof forming nucleic acid duplexes) and the like.

The terms “specific-binding molecule” and “affinity molecule” are usedinterchangeably herein and refer to a molecule that will selectivelybind, through chemical or physical means to a detectable substancepresent in a sample. By “selectively bind” is meant that the moleculebinds preferentially to the target of interest or binds with greateraffinity to the target than to other molecules. For example, an antibodywill selectively bind to the antigen against which it was raised; A DNAmolecule will bind to a substantially complementary sequence and not tounrelated sequences. The affinity molecule can comprise any molecule, orportion of any molecule, that is capable of being linked to asemiconductor nanocrystal and that, when so linked, is capable ofrecognizing specifically a detectable substance. Such affinity moleculesinclude, by way of example, such classes of substances as antibodies, asdefined below, monomeric or polymeric nucleic acids, aptamers, proteins,polysaccharides, sugars, and the like. See,. e.g., Haugland, “Handbookof Fluorescent Probes and Research Chemicals” (Sixth Edition), and anyof the molecules capable of forming a binding pair as described above.

A “semiconductor nanocrystal conjugate” is a semiconductor nanocrystalwhich is linked to or associated with a specific-binding molecule, asdefined above. A “semiconductor nanocrystal conjugate” includes, forexample, a semiconductor nanocrystal linked or otherwise associated,through the coat, to a member of a “binding pair” or a “specific-bindingmolecule” that will selectively bind to a detectable substance presentin a sample, e.g., a biological sample as defined herein. The firstmember of the binding pair linked to the semiconductor nanocrystal cancomprise any molecule, or portion of any molecule, that is capable ofbeing linked to a semiconductor nanocrystal and that, when so linked, iscapable of recognizing specifically the second member of the bindingpair.

The term “antibody” as used herein includes antibodies obtained fromboth polyclonal and monoclonal preparations, as well as, the following:hybrid (chimeric) antibody molecules (see, for example, Winter et al.(1991) Nature 349:293-299; and U.S. Pat. No. 4,816,567); F(ab′)2 andF(ab) fragments; Fv molecules (noncovalent heterodimers, see, forexample, Inbar et al. (1972) Proc Natl Acad Sci USA 69:2659-2662; andEhrlich et al. (1980) Biochem 19:4091-4096); single-chain Fv molecules(sFv) (see, for example, Huston et al. (1988) Proc Natl Acad Sci USA85:5879-5883); dimeric and trimeric antibody fragment constructs;minibodies (see, e.g., Pack et al. (1992) Biochem 31:1579-1584; Cumberet al. (1992) J Immunology 149B: 120-126); humanized antibody molecules(see, for example, Riechmann et al. (1988) Nature 332:323-327; Verhoeyanet al. (1988) Science 239:1534-1536; and U.K. Patent Publication No. GB2,276,169, published 21 Sep. 1994); and, any functional fragmentsobtained from such molecules, wherein such fragments retainspecific-binding properties of the parent antibody molecule.

As used herein, the term “monoclonal antibody” refers to an antibodycomposition having a homogeneous antibody population. The term is notlimited regarding the species or source of the antibody, nor is itintended to be limited by the manner in which it is made. Thus, the termencompasses antibodies obtained from murine hybridomas, as well as humanmonoclonal antibodies obtained using human rather than murinehybridomas. See, e.g., Cote, et al. Monclonal Antibodies and CancerTherapy, Alan R. Liss, 1985, p. 77.

A semiconductor nanocrystal is “linked” or “conjugated” to, or“associated” with, a specific-binding molecule or member of a bindingpair when the semiconductor nanocrystal is chemically coupled to, orassociated with the specific-binding molecule. Thus, these terms intendthat the semiconductor nanocrystal may either be directly linked to thespecific-binding molecule or may be linked via a linker moiety, such asvia a chemical linker described below. The terms indicate items that arephysically linked by, for example, covalent chemical bonds, physicalforces such van der Waals or hydrophobic interactions, encapsulation,embedding, or the like. As an example without limiting the scope of theinvention, nanocrystals can be conjugated to molecules that can interactphysically with biological compounds such as cells, proteins, nucleicacids, subcellular organelles and other subcellular components. Forexample, nanocrystals can be associated with biotin which can bind tothe proteins, avidin and streptavidin. Also, nanocrystals can beassociated with molecules that bind nonspecifically orsequence-specifically to nucleic acids (DNA RNA). As examples withoutlimiting the scope of the invention, such molecules include smallmolecules that bind to the minor groove of DNA (for reviews, seeGeierstanger and Wemmer (1995) Ann. Rev. Biophys. Biomol. Strut.24:463-493; and Baguley (1982) Mol. Cell. Biochem 43:167-181), smallmolecules that form adducts with DNA and RNA (e.g. CC-1065, seeHenderson and Hurley (1996) J. Mol. Recognit. 9:75-87; aflatoxin, seeGarner (1998) Mutat. Res. 402:67-75; cisplatin, see Leng and Brabec(1994) IARC Sci. Publ. 125:339-348), molecules that intercalate betweenthe base pairs of DNA (e.g. methidium, propidium, ethidium, porphyrins,etc., for a review see Bailly et al. J. Mol. Recognit. 5:155-171),radiomimetic DNA damaging agents such as bleomycin, neocarzinostatin andother enediynes (for a review, see Povirk (1996) Mutat. Res. 355:71-89),and metal complexes that bind and/or damage nucleic acids throughoxidation (e.g. Cu-phenanthroline, see Perrin et al. (1996) Prog.Nucleic Acid Res. Mol. Biol. 52:123-151; Ru(II) and Os(II) complexes,see Moucheron et al. (1997) J. Photochem. Photobiol. B 40:91-106;chemical and photochemical probes of DNA, see Nielsen (1990) J. Mol.Recognit. 3:1-25.

As used herein, a “biological sample” refers to a sample of isolatedcells, tissue or fluid, including but not limited to, for example,plasma, serum, spinal fluid, semen, lymph fluid, the external sectionsof the skin, respiratory, intestinal, and genitourinary tracts, tears,saliva, milk, blood cells, tumors, organs, and also samples of in vitrocell culture constituents (including but not limited to conditionedmedium resulting from the growth of cells in cell culture medium,putatively virally infected cells, recombinant cells, and cellcomponents).

A “small molecule” is defined as including an organic or inorganiccompound either synthesized in the laboratory or found in nature.Typically, a small molecule is characterized in that it contains severalcarbon-carbon bonds, and has a molecular weight of less than 1500grams/Mol.

A “biomolecule” is a synthetic or naturally occurring molecule, such asa protein, amino acid, nucleic acid, nucleotide, carbohydrate, sugar,lipid and the like.

A “homogeneous assay” is one that is performed without transfer,separation or washing steps. Thus, for example, a homogeneous HTS assayinvolves the addition of reagents to a vessel, e.g., a test tube orsample well, followed by the detection of the results from thatparticular well. A homogeneous HTS assay can be performed in thesolution in the test tube or well, on the surface of the test tube orwell, on beads or cells which are placed into the test tube or the well,or the like. The detection system typically used is a fluorescence,chemiluminescence, or scintillation detection system.

The term “multiplexing” is used herein to include conducting an assay orother analytical method in which multiple analytes or biological statescan be detected simultaneously by using more than one detectable label,each of which emits at a distinct wavelength, with a distinct intensity,with a distinct FWHM, with a distinct fluorescence lifetime, or anycombination thereof. Preferably, each detectable label is linked to oneof a plurality of first members of binding pairs each of which firstmembers is capable of binding to a distinct corresponding second memberof the binding pair. A multiplexed method using semiconductornanocrystals having distinct emission spectra can be used to detectsimultaneously in the range of 2 to 1,000,000, preferably in the rangeof 2 to 10,000, more preferably in the range of 2 to 100, or any integerbetween these ranges, and even more preferably in the range of up to 10to 20, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20, of analytes, biological compounds or biological states.Multiplexing also includes assays or methods in which the combination ofmore than one semiconductor nanocrystal having distinct emission spectracan be used to detect a single analyte.

The term “barcode” as used herein refers to one or more sizes, sizedistributions, compositions, or any combination thereof, ofsemiconductor nanocrystals. Each size, size distribution and/orcomposition of semiconductor nanocrystals has a characteristic emissionspectrum, e.g., wavelength, intensity, FWHM, and/or fluorescentlifetime. In addition to the ability to tune the emission energy bycontrolling the size of the particular semiconductor nanocrystal, theintensities of that particular emission observed at a specificwavelength are also capable of being varied, thus increasing thepotential information density provided by the semiconductor nanocrystalbarcode system. In preferred embodiments, 2-15 different intensities maybe achieved for a particular emission at a desired wavelength, however,one of ordinary skill in the art will realize that more than fifteendifferent intensities may be achieved, depending upon the particularapplication of interest. For the purposes of the present invention,different intensities may be achieved by varying the concentrations ofthe particular size semiconductor nanocrystal attached to, embeddedwithin or associated with an item, compound or matter of interest. The“barcode” enables the determination of the location or identity of aparticular item, compound or matter of interest. For example,semiconductor nanocrystals can be used to barcode chromosomes, as wellas portions of chromosomes, for spectral karyotyping, as describedfurther below.

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where the event or circumstance occurs and instances in whichit does not. For example, the phrase “optionally overcoated with a shellmaterial” means that the overcoating referred to may or may not bepresent in order to fall within the scope of the invention, and that thedescription includes both the presence and absence of such overcoating.

II. MODES OF CARRYING OUT THE INVENTION

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to specific assay formats,materials or reagents, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments of the invention only, and is notintended to be limiting.

Although a number of compositions and methods similar or equivalent tothose described herein can be used in the practice of the presentinvention, the preferred materials and methods are described herein.

The present invention provides chemical and biological assays which usesemiconductor nanocrystals as detectable luminescent labels to detectthe presence or amount of one or more molecules, as well as to detectbiological interactions, biological processes, alterations in biologicalprocesses, or alterations in the structure of a chemical or biologicalcompound.

Semiconductor nanocrystals demonstrate quantum confinement effects intheir luminescent properties. When semiconductor nanocrystals areilluminated with a primary energy source, a secondary emission of energyoccurs of a frequency that corresponds to the bandgap of thesemiconductor material used in the semiconductor nanocrystal. In quantumconfined particles, the bandgap energy is a function of the size and/orcomposition of the nanocrystal. A mixed population of semiconductornanocrystals of various sizes and/or compositions can be excitedsimultaneously using a single wavelength of light and the detectableluminescence can be engineered to occur at a plurality of wavelengths.The luminescent emission is related to the size and/or the compositionof the constituent semiconductor nanocrystals of the population.Furthermore, semiconductor nanocrystals can be made highly luminescentthrough the use of a shell material which efficiently encapsulates thesurface of the semiconductor nanocrystal core. A “core/shell”semiconductor nanocrystal has a high quantum efficiency andsignificantly improved photochemical stability. The surface of thecore/shell semiconductor nanocrystal can be modified to producesemiconductor nanocrystals that can be coupled to a variety ofbiological molecules or substrates by techniques described in, forexample, Bruchez et. al. (1998) Science 281:2013-2016, Chan et. al.(1998) Science 281:2016-2018, Bruchez “Luminescent SemiconductorNanocrystals: Intermittent Behavior and use as Fluorescent BiologicalProbes” (1998) Doctoral dissertation, University of California,Berkeley, Mikulec “Semiconductor Nanocrystal Colloids Manganese DopedCadmium Selenide, (Core)Shell Composites for Biological Labeling, andHighly Fluorescent Cadmium Telluride” (1999) Doctoral dissertation,Massachusetts Institute of Technology, and described further below.

It is readily apparent that semiconductor nanocrystals can be used todetect or track a single target. Additionally, a population ofsemiconductor nanocrystals may be used for either simultaneous detectionof multiple targets or to detect particular compounds and/or items ofinterest in, e.g., a library of compounds.

For example, compositions of semiconductor nanocrystals comprising oneor more particle size distributions having characteristic spectralemissions may be used as “barcodes” in assays to either track thelocation or source of a particular item of interest or to identify aparticular item of interest. The semiconductor nanocrystals used in sucha “barcoding” scheme can be tuned to a desired wavelength to produce acharacteristic spectral emission by changing the composition and size,or size distribution, of the semiconductor nanocrystal. Additionally,the intensity of the emission at a particular characteristic wavelengthcan also be varied, thus enabling the use of binary or higher orderencoding schemes. The information encoded by the semiconductornanocrystals can be spectroscopically decoded, thus providing thelocation and/or identity of the particular item or component ofinterest.

The ability to use semiconductor nanocrystals in order to detectmultiple targets results from their unique characteristics.Semiconductor nanocrystals have radii that are smaller than the bulkexciton Bohr radius and constitute a class of materials intermediatebetween molecular and bulk forms of matter. Quantum confinement of boththe electron and hole in all three dimensions leads to an increase inthe effective band gap of the material with decreasing crystallite size.Consequently, both the optical absorption and emission of semiconductornanocrystals shift to the blue (higher energies). Upon exposure to aprimary light source, each semiconductor nanocrystal distribution iscapable of emitting energy in narrow spectral linewidths, as narrow as12 nm to 60 nm FWHM, and with a symmetric, nearly Gaussian line shape,thus providing an easy way to identify a particular semiconductornanocrystal. As one of ordinary skill in the art will realize, thelinewidths are dependent on the size heterogeneity, i.e.,monodispersity, of the semiconductor nanocrystals in each preparation.Single semiconductor nanocrystal complexes have been observed to haveFWHM as narrow as 12 nm to 15 nm. In addition, semiconductor nanocrystaldistributions with larger linewidths in the range of 35 nm to 60 nm canbe readily made and have the same physical characteristics assemiconductor nanocrystals with narrower linewidths.

Semiconductor nanocrystals can be used to detect the presence and/oramount of a biological moiety, e.g., a biological target analyte; thestructure, composition, and conformation of a biological molecule; thelocalization of a biological moiety, e.g., a biological target analytein an environment; interactions of biological molecules; alterations instructures of biological compounds; and/or alterations in biologicalprocesses.

Thus, it is readily apparent that semiconductor nanocrystals find use ina variety of assays where other, less reliable, labeling methods havetypically been used, including, without limitation, fluorescencemicroscopy, histology, cytology, pathology, flow cytometry, FISH andother nucleic acid hybridization assays, signal amplification assays,DNA and protein sequencing, immunoassays such as competitive bindingassays and ELISAs, immunohistochemical analysis, protein and nucleicacid separation, homogeneous assays, multiplexing, high throughputscreening, chromosome karyotyping, and the like.

Production of Semiconductor Nanocrystals

Semiconductor nanocrystals for use in the subject methods are made usingtechniques known in the art. See, e.g., U.S. Pat. Nos. 6,048,616;5,990,479; 5,690,807; 5,505,928; 5,262,357 (all of which areincorporated herein in their entireties); as well as PCT Publication No.99/26299 (published May 27, 1999). In particular, exemplary materialsfor use as semiconductor nanocrystals in the biological and chemicalassays of the present invention include, but are not limited to thosedescribed above, including group II-VI, III-V and group IVsemiconductors such as ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, MgS, MgSe,MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, GaN, GaP, GaAs,GaSb, InP, InAs, InSb, AlS, AlP, AlSb, PbS, PbSe, Ge and Si and ternaryand quaternary mixtures thereof. The semiconductor nanocrystals arecharacterized by their uniform nanometer size.

As discussed above, the selection of the composition of thesemiconductor nanocrystal, as well as the size of the semiconductornanocrystal, affects the characteristic spectral emission wavelength ofthe semiconductor nanocrystal. Thus, as one of ordinary skill in the artwill realize, a particular composition of a semiconductor nanocrystal aslisted above will be selected based upon the spectral region beingmonitored. For example, semiconductor nanocrystals that emit energy inthe visible range include, but are not limited to, CdS, CdSe, CdTe,ZnSe, ZnTe, GaP, and GaAs. Semiconductor nanocrystals that emit energyin the near IR range include, but are not limited to, InP, InAs, InSb,PbS, and PbSe. Finally, semiconductor nanocrystals that emit energy inthe blue to near-ultraviolet include, but are not limited to, ZnS andGaN.

For any particular composition selected for the semiconductornanocrystals to be used in the inventive methods, it is possible to tunethe emission to a desired wavelength by controlling the size of theparticular composition of the semiconductor nanocrystal. In preferredembodiments, 5-20 discrete emissions (five to twenty different sizepopulations or distributions distinguishable from one another), morepreferably 10-15 discrete emissions, are obtained for any particularcomposition, although one of ordinary skill in the art will realize thatfewer than five emissions and more than twenty emissions could be useddepending on the monodispersity of the semiconductor nanocrystalparticles. If high information density is required, and thus a greaternumber of distinct emissions, the nanocrystals are preferablysubstantially monodisperse within the size range given above.

As explained above, “monodisperse,” as that term is used herein, means acolloidal system in which the suspended particles have substantiallyidentical size and shape. In preferred embodiments for high informationdensity applications, monodisperse particles deviate less than 10% rmsin diameter, and preferably less than 5%. Monodisperse semiconductornanocrystals have been described in detail in Murray et al. (1993) J.Am. Chem. Soc. 115:8706, and in Murray, “Synthesis and Characterizationof II-VI Quantum Dots and Their Assembly into 3-D Quantum DotSuperlattices,” (1995) Doctoral dissertation, Massachusetts Institute ofTechnology, which are hereby incorporated by reference in theirentireties. One of ordinary skill in the art will also realize that thenumber of discrete emissions that can be distinctly observed for a givencomposition depends not only upon the monodispersity of the particles,but also on the deconvolution techniques employed. Semiconductornanocrystals, unlike dye molecules, can be easily modeled as Gaussiansand therefore are more easily and more accurately deconvoluted.

However, for some applications high information density will not berequired and it may be more economically attractive to use morepolydisperse particles. Thus, for applications that do not require highinformation density, the linewidth of the emission may be in the rangeof 40-60 nm.

In a particularly preferred embodiment, the surface of the semiconductornanocrystal is also modified to enhance the efficiency of the emissions,by adding an overcoating layer to the semiconductor nanocrystal. Theovercoating layer is particularly preferred because at the surface ofthe semiconductor nanocrystal, surface defects can result in traps forelectrons or holes that degrade the electrical and optical properties ofthe semiconductor nanocrystal. An insulating layer at the surface of thesemiconductor nanocrystal provides an atomically abrupt jump in thechemical potential at the interface that eliminates energy states thatcan serve as traps for the electrons and holes. This results in higherefficiency in the luminescent process.

Suitable materials for the overcoating layer include semiconductormaterials having a higher bandgap energy than the semiconductornanocrystal core. In addition to having a bandgap energy greater thanthe semiconductor nanocrystal core, suitable materials for theovercoating layer should have good conduction and valence band offsetwith respect to the core semiconductor nanocrystal. Thus, the conductionband is desirably higher and the valence band is desirably lower thanthose of the core semiconductor nanocrystal. For semiconductornanocrystal cores that emit energy in the visible (e.g., CdS, CdSe,CdTe, ZnSe, ZnTe, GaP, GaAs) or near IR (e.g., InP, InAs, InSb, PbS,PbSe), a material that has a bandgap energy in the ultraviolet regionsmay be used. Exemplary materials include ZnS, GaN, and magnesiumchalcogenides, e.g., MgS, MgSe, and MgTe. For a semiconductornanocrystal core that emits in the near IR, materials having a bandgapenergy in the visible, such as CdS or CdSe, may also be used. Thepreparation of a coated semiconductor nanocrystal may be found in, e.g.,Dabbousi et al. (1997) J. Phys. Chem. B 101:9463) and Kuno et al. (1997)J. Phys. Chem. 106:9869.

Most semiconductor nanocrystals are prepared in coordinating solvent,such as trioctylphosphine oxide (TOPO) and trioctyl phosphine (TOP)resulting in the formation of a passivating organic layer on thenanocrystal surface comprised of the organic solvent. This layer ispresent on semiconductor nanocrystals containing an overcoating andthose that do not contain an overcoating. Thus, either of these classesof passivated semiconductor nanocrystals is readily soluble in organicsolvents, such as toluene, chloroform and hexane. As one of ordinaryskill in the art will realize, these functional moieties may be readilydisplaced or modified to provide an outer coating that renders thesemiconductor nanocrystals suitable for use as the detectable labels ofthe present invention, as described further below. Furthermore, basedupon the desired application, a portion of the semiconductor nanocrystalfunctionality, or the entire surface of the semiconductor nanocrystalfunctionality may be modified by a displacement reaction, based upon thedesired use therefor.

After selection of the composition of semiconductor nanocrystal for thedesired range of spectral emission and selection of a desired surfacefunctionalization compatible with the system of interest, it may also bedesirable to select the minimum number of semiconductor nanocrystalsneeded to observe a distinct and unique spectral emission of sufficientintensity for spectral identification. Selection criteria important indetermining the minimum number of semiconductor nanocrystals needed toobserve a distinct and unique spectral emission of sufficient intensityinclude providing a sufficient number of semiconductor nanocrystals thatare bright (i.e., that emit light versus those that are dark) andproviding a sufficient number of semiconductor nanocrystals to averageout over the blinking effect observed in single semiconductornanocrystal emissions. Nirmal et al., (1996) Nature 383:802.

For example, eight or more semiconductor nanocrystals of a particularcomposition and particle size distribution can be provided. If, forexample, the desired method of use utilizes three different particlesize distributions of a particular composition, eight of each of thethree different particle size distributions of a semiconductornanocrystal is used, in order to observe sufficiently intense spectralemissions from each to provide reliable information regarding thelocation or identity of a particular analyte of interest. One ofordinary skill in the art will realize, however, that fewer than eightsemiconductor nanocrystals of a particular composition and particle sizedistribution may be utilized provided that a unique spectral emission ofsufficient intensity is observed, as determined by the selectioncriteria set forth above.

The above method can be used to prepare separate populations ofsemiconductor nanocrystals, wherein each population exhibits a differentcharacteristic photoluminescence spectrum. Each of a plurality ofpopulations of semiconductor nanocrystals can be conjugated to distinctfirst members of binding pairs for use in a multiplexed assay oranalytical method in which each of a plurality of corresponding secondmembers of the binding pairs can be detected simultaneously.

The narrow spectral linewidths and nearly gaussian symmetricallineshapes lacking a tailing region observed for the emission spectra ofnanocrystals combined with the tunability of the emission wavelengths ofnanocrystals allows high spectral resolution in a system with multiplenanocrystals. In theory up to 10-20 or more different-sized nanocrystalsor different size distributions of monodisperse populations ofnanocrystals from different preparations of nanocrystals, with eachsample having a different emission spectrum, can be used simultaneouslyin one system, i.e., multiplexing, with the overlapping spectra easilyresolved using techniques well known in the art, e.g., optically with orwithout the use of deconvolution software.

As discussed previously, the ability of the semiconductor nanocrystalsto produce discrete optical transitions, along with the ability to varythe intensity of these optical transitions, enables the development of aversatile and dense encoding scheme. The characteristic emissionsproduced by one or more sizes of semiconductor nanocrystals attached to,associated with, or embedded within a particular support, compound ormatter enables the identification of the analyte of interest and/or itslocation. For example, by providing N sizes of semiconductornanocrystals (each having a discrete optical transition), each having Mdistinguishable states resulting from the absence of the semiconductornanocrystal, or from different intensities resulting from a particulardiscrete optical transition, M^(n) different states can be uniquelydefined. In the case wherein M is 2, in which the two states could bethe presence or absence of the semiconductor nanocrystal, the encodingscheme would thus be defined by a base 2 or binary code. In the casewherein M is 3, in which the three states could be the presence of asemiconductor nanocrystal at two distinguishable intensities and itsabsence, the encoding scheme would be defined by a base 3 code. Herein,such base M codes wherein M is greater than 2 are termed higher ordercodes. The advantage of higher order codes over a binary order code isthat fewer identifiers are required to encode the same quantity ofinformation.

As one of ordinary skill in the art will realize, the ability to developa higher order encoding system is dependent upon the number of differentintensities capable of detection by both the hardware and the softwareutilized in the decoding system. In particularly preferred embodiments,each discrete emission or color, is capable of being detectable at twoto twenty different intensities. In a particularly preferred embodimentwherein ten different intensities are available, it is possible toemploy a base 11 code comprising the absence of the semiconductornanocrystal, or the detection of the semiconductor nanocrystal at 10different intensities.

Clearly, the advantages of the semiconductor nanocrystals, namely theability to observe discrete optical transitions at a plurality ofintensities, provides a powerful and dense encoding scheme that can beemployed in a variety of disciplines. In general, one or moresemiconductor nanocrystals may act as a barcode, wherein each of the oneor more semiconductor nanocrystals produces a distinct emissionsspectrum. These characteristic emissions can be observed as colors, ifin the visible region of the spectrum, or may also be decoded to provideinformation about the particular wavelength at which the discretetransition is observed. Likewise, for semiconductor nanocrystalsproducing emissions in the infrared or ultraviolet regions, thecharacteristic wavelengths that the discrete optical transitions occurat provide information about the identity of the particularsemiconductor nanocrystal, and hence about the identity of or locationof the analyte of interest.

The color of light produced by a particular size, size distributionand/or composition of a semiconductor nanocrystal can be readilycalculated or measured by methods which will be apparent to thoseskilled in the art. As an example of these measurement techniques, thebandgaps for nanocrystals of CdSe of sizes ranging from 12 Å to 115 Åare given in Murray et al. (1993) J. Am. Chem. Soc. 115:8706. Thesetechniques allow ready calculation of an appropriate size, sizedistribution and/or composition of semiconductor nanocrystals and choiceof excitation light source to produce a nanocrystal capable of emittinglight device of any desired wavelength.

An example of a specific system for automated detection for use with thepresent methods includes, but is not limited to, an imaging schemecomprising an excitation source, a monochromator (or any device capableof spectrally resolving the image, or a set of narrow band filters) anda detector array. In one embodiment, the apparatus consists of a blue orUV source of light, of a wavelength shorter than that of theluminescence detected. This may be a broadband UV light source, such asa deuterium lamp with a filter in front; the output of a white lightsource such as a xenon lamp or a deuterium lamp after passing through amonochromator to extract out the desired wavelengths; or any of a numberof continuous wave (cw) gas lasers, including but not limited to any ofthe Argon Ion laser lines (457, 488, 514, etc. nm), a HeCd laser; solidstate diode lasers in the blue such as GaN and GaAs (doubled) basedlasers or the doubled or tripled output of YAG or YLF based lasers; orany of the pulsed lasers with output in the blue, to name a few.

The luminescence from the dots may be passed through an imagingsubtracting double monochromator (or two single monochromators with thesecond one reversed from the first), for example, consisting of twogratings or prisms and a slit between the two gratings or prisms. Themonochromators or gratings or prisms can also be replaced with acomputer controlled color filter wheel where each filter is a narrowband filter centered at the wavelength of emission of one of the dots.The monochromator assembly has more flexibility because any color can bechosen as the center wavelength. Furthermore, a CCD camera or some othertwo dimensional detector records the images, and software color codesthat image to the wavelength chosen above. The system then moves thegratings to a new color and repeats the process. As a result of thisprocess, a set of images of the same spatial region is obtained and eachis color-coded to a particular wavelength that is needed to analyze thedata rapidly.

In another embodiment, the apparatus is a scanning system as opposed tothe above imaging scheme. In a scanning scheme, the sample to beanalyzed is scanned with respect to a microscope objective. Theluminescence is put through a single monochromator or a grating or prismto spectrally resolve the colors. The detector is a diode array thatthen records the colors that are emitted at a particular spatialposition. The software then ultimately recreates the scanned image anddecodes it.

Production of Semiconductor Nanocrystal Conjugates

The present invention uses a composition comprising semiconductornanocrystals associated with a specific-binding molecule or affinitymolecule, such that the composition can detect the presence and/oramounts of biological and chemical compounds, detect interactions inbiological systems, detect biological processes, detect alterations inbiological processes, or detect alterations in the structure ofbiological compounds. Without limitation, semiconductor nanocrystalconjugates comprise any molecule or molecular complex, linked to asemiconductor nanocrystal, that can interact with a biological target,to detect biological processes, or reactions, as well as alterbiological molecules or processes. Preferably, the molecules ormolecular complexes or conjugates physically interact with a biologicalcompound. Preferably, the interactions are specific. The interactionscan be, but are not limited to, covalent, noncovalent, hydrophobic,hydrophilic, electrostatic, van der Waals, or magnetic. Preferably,these molecules are small molecules, proteins, or nucleic acids orcombinations thereof.

Semiconductor nanocrystal conjugates can be made using techniques knownin the art. For example, moieties such as TOPO and TOP, generally usedin the production of semiconductor nanocrystals, as well as othermoieties, may be readily displaced and replaced with other functionalmoieties, including, but not limited to carboxylic acids, amines,aldehydes, and styrene to name a few. One of ordinary skill in the artwill realize that factors relevant to the success of a particulardisplacement reaction include the concentration of the replacementmoiety, temperature and reactivity. Thus, for the purposes of thepresent invention, any functional moiety may be utilized that is capableof displacing an existing functional moiety to provide a semiconductornanocrystal with a modified functionality for a specific use.

The ability to utilize a general displacement reaction to modifyselectively the surface functionality of the semiconductor nanocrystalsenables functionalization for specific uses. For example, becausedetection of biological compounds is most preferably carried out inaqueous media, a preferred embodiment of the present invention utilizessemiconductor nanocrystals that are solubilized in water. In the case ofwater-soluble semiconductor nanocrystals, the outer layer includes acompound having at least one linking moiety that attaches to the surfaceof the particle and that terminates in at least one hydrophilic moiety.The linking and hydrophilic moieties are spanned by a hydrophobic regionsufficient to prevent charge transfer across the region. The hydrophobicregion also provides a “pseudo-hydrophobic” environment for thenanocrystal and thereby shields it from aqueous surroundings. Thehydrophilic moiety may be a polar or charged (positive or negative)group. The polarity or charge of the group provides the necessaryhydrophilic interactions with water to provide stable solutions orsuspensions of the semiconductor nanocrystal. Exemplary hydrophilicgroups include polar groups such as hydroxides (—OH), amines,polyethers, such as polyethylene glycol and the like, as well as chargedgroups, such as carboxylates (—CO²⁻), sulfonates (SO³⁻), phosphates(—PO₄ ²⁻) and —PO₃ ²⁻), nitrates, ammonium salts (—NH⁴⁺), and the like.A water-solubilizing layer is found at the outer surface of theovercoating layer. Methods for rendering semiconductor nanocrystalswater-soluble are known in the art and described in, e.g., InternationalPublication No. WO 00/17655, published Mar. 30, 2000.

The affinity for the nanocrystal surface promotes coordination of thelinking moiety to the semiconductor nanocrystal outer surface and themoiety with affinity for the aqueous medium stabilizes the semiconductornanocrystal suspension.

A displacement reaction may be employed to modify the semiconductornanocrystal to improve the solubility in a particular organic solvent.For example, if it is desired to associate the semiconductornanocrystals with a particular solvent or liquid, such as pyridine, thesurface can be specifically modified with pyridine or pyridine-likemoieties to ensure solvation.

The surface layer may also be modified by displacement to render thesemiconductor nanocrystal reactive for a particular coupling reaction.For example, displacement of TOPO moieties with a group containing acarboxylic acid moiety enables the reaction of the modifiedsemiconductor nanocrystals with amine containing moieties (commonlyfound on solid support units) to provide an amide linkage. Additionalmodifications can also be made such that the semiconductor nanocrystalcan be associated with almost any solid support. A solid support, forthe purposes of this invention, is defined as an insoluble material towhich compounds are attached during a synthesis sequence, screening,immunoassays, etc. The use of a solid support is particularlyadvantageous for the synthesis of libraries because the isolation ofsupport-bound reaction products can be accomplished simply by washingaway reagents from the support-bound material and therefore the reactioncan be driven to completion by the use of excess reagents.

A solid support can be any material that is an insoluble matrix and canhave a rigid or semi-rigid surface. Exemplary solid supports include butare not limited to pellets, disks, capillaries, hollow fibers, needles,pins, solid fibers, cellulose beads, pore-glass beads, silica gels,polystyrene beads optionally cross-linked with divinylbenzene, graftedco-poly beads, polyacrylamide beads, latex beads, dimethylacrylamidebeads optionally crosslinked with N—N′-bis-acryloylethylenediamine, andglass particles coated with a hydrophobic polymer.

For example, the semiconductor nanocrystals of the present invention canreadily be functionalized to create styrene or acrylate moieties, thusenabling the incorporation of the semiconductor nanocrystals intopolystyrene, polyacrylate or other polymers such as polyimide,polyacrylamide, polyethylene, polyvinyl, polydiacetylene,polyphenylene-vinylene, polypeptide, polysaccharide, polysulfone,polypyrrole, polyimidazole, polythiophene, polyether, epoxies, silicaglass, silica gel, siloxane, polyphosphate, hydrogel, agarose,cellulose, and the like.

For a detailed description of these linking reactions, see, e.g., U.S.Pat. No. 5,990,479; Bruchez et. al. (1998) Science 281:2013-2016., Chanet. al. (1998) Science 281:2016-2018, Bruchez “Luminescent SemiconductorNanocrystals: Intermittent Behavior and use as Fluorescent BiologicalProbes” (1998) Doctoral dissertation, University of California,Berkeley, and Mikulec “Semiconductor Nanocrystal Colloids: ManganeseDoped Cadmium Selenide, (Core)Shell Composites for Biological Labeling,and Highly Fluorescent Cadmium Telluride” (1999) Doctoral dissertation,Massachusetts Institute of Technology.

Semiconductor Nanocrystals as Detection Reagents in Immunoassays

In one embodiment of the invention, immunoassays, such as immunosorbentassays, are provided in which semiconductor nanocrystal conjugates areused as the detection reagents. A Qdot™ immunosorbent assay (QISA) hasseveral advantages over current immunosorbent assays including, but notlimited to, simultaneous multicolor detection and, hence, multipleanalyte detection, no requirement for enzyme development, increasedphotostability over alternative fluorophores thereby allowing increaseddetection sensitivity by virtue of the ability to monitor the signalover a long period of time, increased sensitivity over enzyme-baseddetection systems.

Semiconductor nanocrystals of varying core sizes (10-150 Å), compositionand/or size distribution are conjugated to specific-binding moleculeswhich bind specifically to an analyte of interest. Any specificanti-analyte can be used, for example, an antibody, an immunoreactivefragment of an antibody, and the like. Preferably, the anti-analyte isan antibody. The semiconductor nanocrystal conjugates are used in animmunosorbent assay to detect any analyte for which a specific-bindingagent exists.

More specifically, the specific-binding molecule may be derived frompolyclonal or monoclonal antibody preparations, may be a human antibody,or may be a hybrid or chimeric antibody, such as a humanized antibody,an altered antibody, F(ab′)₂ fragments, F(ab) fragments, Fv fragments, asingle-domain antibody, a dimeric or trimeric antibody fragmentconstruct, a minibody, or functional fragments thereof which bind to theanalyte of interest. Antibodies are produced using techniques well knownto those of skill in the art and disclosed in, for example, U.S. Pat.Nos. 4,011,308; 4,722,890; 4,016,043; 3,876,504; 3,770,380; and4,372,745,

For example, polyclonal antibodies are generated by immunizing asuitable animal, such as a mouse, rat, rabbit, sheep or goat, with anantigen of interest. In order to enhance immunogenicity, the antigen canbe linked to a carrier prior to immunization. Such carriers are wellknown to those of ordinary skill in the art.

Immunization is generally performed by mixing or emulsifying the antigenin saline, preferably in an adjuvant such as Freund's complete adjuvant,and injecting the mixture or emulsion parenterally (generallysubcutaneously or intramuscularly). The animal is generally boosted 2-6weeks later with one or more injections of the antigen in saline,preferably using Freund's incomplete adjuvant. Antibodies may also begenerated by in vitro immunization, using methods known in the art.Polyclonal antiserum is then obtained from the immunized animal.

Monoclonal antibodies are generally prepared using the method of Kohlerand Milstein (1975) Nature 256:495-497, or a modification thereof.Typically, a mouse or rat is immunized as described above. However,rather than bleeding the animal to extract serum, the spleen (andoptionally several large lymph nodes) is removed and dissociated intosingle cells. If desired, the spleen cells may be screened (afterremoval of nonspecifically adherent cells) by applying a cell suspensionto a plate or well coated with the antigen. B-cells, expressingmembrane-bound immunoglobulin specific for the antigen, will bind to theplate, and are not rinsed away with the rest of the suspension.Resulting B-cells, or all dissociated spleen cells, are then induced tofuse with myeloma cells to form hybridomas, and are cultured in aselective medium (e.g., hypoxanthine, aminopterin, thymidine medium,“HAT”). The resulting hybridomas are plated by limiting dilution, andare assayed for the production of antibodies which bind specifically tothe immunizing antigen (and which do not bind to unrelated antigens).The selected monoclonal antibody-secreting hybridomas are then culturedeither in vitro (e.g., in tissue culture bottles or hollow fiberreactors), or in vivo (e.g., as ascites in mice).

Human monoclonal antibodies are obtained by using human rather thanmurine hybridomas. See, e.g., Cote, et al. Monclonal Antibodies andCancer Therapy, Alan R. Liss, 1985, p. 77

Monoclonal antibodies or portions thereof may be identified by firstscreening a B-cell cDNA library for DNA molecules that encode antibodiesthat specifically bind to p185, according to the method generally setforth by Huse et al. (1989) Science 246:1275-1281. The DNA molecule maythen be cloned and amplified to obtain sequences that encode theantibody (or binding domain) of the desired specificity.

As explained above, antibody fragments which retain the ability torecognize the analyte of interest, will also find use in the subjectimmunoassays. A number of antibody fragments are known in the art whichcomprise antigen-binding sites capable of exhibiting immunologicalbinding properties of an intact antibody molecule. For example,functional antibody fragments can be produced by cleaving a constantregion, not responsible for antigen binding, from the antibody molecule,using e.g., pepsin, to produce F(ab′)₂ fragments. These fragments willcontain two antigen binding sites, but lack a portion of the constantregion from each of the heavy chains. Similarly, if desired, Fabfragments, comprising a single antigen binding site, can be produced,e.g., by digestion of polyclonal or monoclonal antibodies with papain.Functional fragments, including only the variable regions of the heavyand light chains, can also be produced, using standard techniques suchas recombinant production or preferential proteolytic cleavage ofimmunoglobulin molecules. These fragments are known as F_(V). See, e.g.,Inbar et al. (1972) Proc. Nat. Acad. Sci. USA 69:2659-2662; Hochman etal. (1976) Biochem 15:2706-2710; and Ehrlich et al. (1980) Biochem19:4091-4096.

A single-chain Fv (“sFv” or “scFv”) polypeptide is a covalently linkedV_(H)-V_(L) heterodimer which is expressed from a gene fusion includingV_(H)- and V_(L)-encoding genes linked by a peptide-encoding linker.Huston et al. (1988) Proc. Nat. Acad. Sci. USA 85:5879-5883. A number ofmethods have been described to discern and develop chemical structures(linkers) for converting the naturally aggregated, but chemicallyseparated, light and heavy polypeptide chains from an antibody V regioninto an sFv molecule which will fold into a three dimensional structuresubstantially similar to the structure of an antigen-binding site. See,e.g., U.S. Pat. Nos. 5,091,513, 5,132,405 and 4,946,778. The sFvmolecules may be produced using methods described in the art. See, e.g.,Huston et al. (1988) Proc. Nat. Acad. Sci. USA 85:5879-5883; U.S. Pat.Nos. 5,091,513, 5,132,405 and 4,946,778. Design criteria includedetermining the appropriate length to span the distance between theC-terminus of one chain and the N-terminus of the other, wherein thelinker is generally formed from small hydrophilic amino acid residuesthat do not tend to coil or form secondary structures. Such methods havebeen described in the art. See, e.g., U.S. Pat. Nos. 5,091,513,5,132,405 and 4,946,778. Suitable linkers generally comprise polypeptidechains of alternating sets of glycine and serine residues, and mayinclude glutamic acid and lysine residues inserted to enhancesolubility.

“Mini-antibodies” or “minibodies” will also find use with the presentinvention. Minibodies are sFv polypeptide chains which includeoligomerization domains at their C-termini, separated from the sFv by ahinge region. Pack et al. (1992) Biochem 31:1579-1584. Theoligomerization domain comprises self-associating α-helices, e.g.,leucine zippers, that can be further stabilized by additional disulfidebonds. The oligomerization domain is designed to be compatible withvectorial folding across a membrane, a process thought to facilitate invivo folding of the polypeptide into a functional binding protein.Generally, minibodies are produced using recombinant methods well knownin the art. See, e.g., Pack et al. (1992) Biochem 31:1579-1584; Cumberet al. (1992) J Immunology 149B: 120-126.

Once produced, the specific-binding molecules described above may beused in immunoassays of varying formats. For example, the antibodies maybe linked to the semiconductor nanocrystal. Alternatively, thesemiconductor nanocrystals may be linked to other affinity molecules,such as strepavidin, that will specifically react with another moleculethat is linked to the analyte of interest.

The analyte of interest can be detected using standard immunoassays suchas competition, direct reaction, or sandwich type assays. Such assaysinclude, but are not limited to, ELISA-like assays (termed QISA herein)and biotin/avidin type assays. The reactions include the semiconductornanocrystals in order to detect the formation of a complex between theantigen and the antibody or antibodies reacted therewith.

The aforementioned assays generally involve separation of unboundantibody in a liquid phase from a solid phase support to which labeledantigen-antibody complexes are bound. Solid supports which can be usedin the methods herein include substrates such as nitrocellulose (e.g.,in membrane or microtiter well form); polyvinylchloride (e.g., sheets ormicrotiter wells); polystyrene latex (e.g., beads or microtiter plates);polyvinylidine fluoride; diazotized paper; nylon membranes; activatedbeads, magnetically responsive beads, and the like.

In one context, a solid support is first reacted with a component thatwill bind to the solid support, i.e., the “solid phase component,” e.g.,an antigen or an antibody, under suitable binding conditions such thatthe component is sufficiently immobilized to the support. Sometimes,immobilization to the support can be enhanced by first coupling theantigen or antibody to a protein with better solid phase-bindingproperties. Suitable coupling proteins include, but are not limited to,macromolecules such as serum albumins including bovine serum albumin(BSA), keyhole limpet hemocyanin, immunoglobulin molecules,thyroglobulin, ovalbumin, and other proteins well known to those skilledin the art. Other reagents that can be used to bind molecules to thesupport include polysaccharides, polylactic acids, polyglycolic acids,polymeric amino acids, amino acid copolymers, and the like. Suchmolecules and methods of coupling these molecules to antigens, are wellknown to those of ordinary skill in the art. See, e.g., Brinkley, M. A.(1992) Bioconjugate Chem. 3:2-13; Hashida et al. (1984) J. Appl.Biochem. 6:56-63; and Anjaneyulu and Staros (1987) International J. ofPeptide and Protein Res. 30:117-124.

After reacting the solid support with the solid phase component, anynonimmobilized solid-phase components are removed from the support bywashing, and the support-bound component is then contacted with abiological sample suspected of containing ligand moieties (e.g.,antibodies toward the immobilized antigens, or antigens towardimmobilized antibodies) under suitable binding conditions. After washingto remove any nonbound ligand, a secondary binder moiety detectablylabeled with the semiconductor nanocrystals described herein is addedunder suitable binding conditions, wherein the secondary binder iscapable of associating selectively with the bound ligand. The presenceof the secondary binder can then be detected using the techniquesdescribed above. Alternatively, an indirect labeling technique can beused wherein the presence of an unlabeled secondary binder moiety isdetected by the specific binding thereto of a detectably labeledtertiary binder moiety.

More particularly, a QISA method can be used, wherein the wells of amicrotiter plate are coated with a selected antigen. A biological samplecontaining or suspected of containing antibodies to the antigen is thenadded to the coated wells. After a period of incubation sufficient toallow antibody binding to the immobilized antigen, the plate(s) can bewashed to remove unbound moieties and a detectably labeled secondarybinding molecule added. The secondary binding molecule is allowed toreact with any captured sample antibodies, the plate washed and thepresence of the secondary binding molecule detected as described above.

Thus, in one particular embodiment, the presence of bound antibodyligands from a biological sample can be readily detected using asecondary binder comprising an antibody directed against the antibodyligands, conjugated to semiconductor nanocrystals. A number ofimmunoglobulin (Ig) molecules are known in the art which can be readilyconjugated to semiconductor nanocrystals as described herein. In otherrelated embodiments, competitive-type QISA techniques can be practicedusing methods known to those skilled in the art.

Assays can also be conducted in solution, such that the antigens andantibodies specific for those proteins form complexes underprecipitating conditions. In one particular embodiment, antigens can beattached to a solid phase particle (e.g., an agarose bead or the like)using coupling techniques known in the art, such as by direct chemicalor indirect coupling. The antigen-coated particle is then contactedunder suitable binding conditions with a biological sample suspected ofcontaining antibodies for the antigen. Cross-linking between boundantibodies causes the formation of particle-antigen-antibody complexaggregates which can be precipitated and separated from the sample usingwashing and/or centrifugation. The reaction mixture can be analyzed todetermine the presence or absence of antibody-antigen complexes usingany of a number of standard methods, such as those immunodiagnosticmethods described above.

In yet a further embodiment, an immunoaffinity matrix can be provided,wherein a polyclonal population of antibodies from a biological samplesuspected of containing a particular antigen is immobilized to asubstrate. In this regard, an initial affinity purification of thesample can be carried out using immobilized antigens. The resultantsample preparation will thus only contain specific antibodies, avoidingpotential nonspecific binding properties in the affinity support. Anumber of methods of immobilizing immunoglobulins (either intact or inspecific fragments) at high yield and good retention of antigen bindingactivity are known in the art. Not being limited by any particularmethod, immobilized protein A or protein G can be used to immobilizeimmunoglobulins.

Accordingly, once the immunoglobulin molecules have been immobilized toprovide an immunoaffinity matrix, semiconductor nanocrystal-labeledproteins are contacted with the bound antibodies under suitable bindingconditions. After any nonspecifically bound antigen has been washed fromthe immunoaffinity support, the presence of bound antigen can bedetermined by assaying for label using methods described above.

Additionally, antibodies raised to particular antigens, rather than theantigens themselves, can be used in the above-described assays in orderto detect the presence of a protein of interest in a given sample. Theseassays are performed essentially as described above and are well knownto those of skill in the art.

In yet further embodiments, semiconductor nanocrystal-antibodyconjugates may be used to probe fixed tissue samples or fixed cellpopulations for specific markers. In this embodiment, prepared cells ortissue are incubated with an antibody which is conjugated to asemiconductor nanocrystal. Semiconductor nanocrystals allow stable,multicolor detection of markers in both cell and tissue samples.

Semiconductor nanocrystal-conjugates (either a single semiconductornanocrystal conjugated to biomolecules or a plurality of semiconductornanocrystals) allow specific, sensitive, photostable detection ofantigens in staining procedures. This offers a clear advantages overcurrently available stains. Additionally the inherent properties ofsemiconductor nanocrystals, i.e., single excitation source, narrow,gaussian spectra and tunability of emission wavelength, mean that manymore colors are resolved than with conventional fluorescent dyes.

More particularly, multiple analysis staining can be performed on atissue sample, blood sample or any sample requiring multiplexed analysisof cellular or extracellular markers. The procedure may be carried outin a two-step reaction whereby a primary antibody is followed by asemiconductor nanocrystal-conjugated antibody or by using an antibody(or other biomolecule) semiconductor nanocrystal conjugate to directlylabel the sample. For example, five (or more with increased spectral useor reduced spectral separation) different populations of semiconductornanocrystals can be synthesized with emission spectra that are spaced at40 nm intervals from, e.g., 490-650 nm. Each spectrally distinctpopulation of semiconductor nanocrystals is conjugated to a differentmolecule which specifically recognizes a biomolecule of interest whichmay or may not be present in the sample to be analyzed. Followingstandard staining protocols, the sample is labeled with thesemiconductor nanocrystals and analyzed for the location and quantity ofthe target molecule. This analysis may be carried out by conventionalfluorescent microscopy techniques or by use of a spectral scanningdevice as described above.

Since many semiconductor nanocrystals can be generated that arespectrally distinct, it is possible to label different items such asantibodies or cDNAs that can then be used to measure the position andquantity of cellular compounds (as described above). The number ofcompounds that can be followed is limited, however, by the number ofspectrally distinct colors that can be made. With CdSe, semiconductornanocrystals and the current synthetic techniques, this is approximately6-7 spectrally distinct colors. If different compounds are notcolocalized in the cell, however, many more semiconductor nanocrystalcolors can be used by taking advantage of the known spatial separationof the targets to be analyzed. For example, no overlap would occurbetween nuclear localized targets and membrane localized targets. Henceorganelle-specific groups of semiconductor nanocrystals can be employedto increase dramatically the number of discernable targets (see Example18 below).

The semiconductor nanocrystals may also be used in competitivemicrosphere filter assays, such as competitive latex immunoassays. Suchassays are described in, e.g., Stave J. W. (1994) Immunoassay forpriority pollutants. Analytica 94 Conference Abstracts p. 339; andBangs, L. B. (1996) Immunological Applications of Microspheres. In TheLatex Course 4196. Traditionally, this assay uses antibody-conjugatedlatex particles to detect industrial chemicals in soil or ground watersamples at parts per billion concentrations. In this application theantibody is conjugated to, e.g., 3 μm microspheres which are caught on 1μm pore filters. A sample is passed through the filter and theimmobilized antibodies catch any antigen present. An enzyme/antigenconjugate is passed through the filter. If the sample contains noantigen then the enzyme/antigen will bind to the free sites on theantibody and added substrate will cause a color change on the filter.Increases in antigen concentration will result in correspondingdecreases in the filter color change.

In the context of the present invention, the detection agent is asemiconductor nanocrystal or a semiconductor nanocrystal-encoded solidconjugate. The conjugate is a molecule that specifically recognizes theanalyte. The antigen conjugates are direct semiconductornanocrystal-antigen conjugates or semiconductor nanocrystal-dyedmicrosphere-conjugates that are small enough to pass through filterpores.

This allows multiple simultaneous detections using a light source forexcitation of the semiconductor nanocrystals and detection of emissions.The detection takes place on the filter or in the filtrate and the assaymay be carried out in a high throughput multiwell environment. Thefilters in this format are opaque to the excitation light and allowdetection of semiconductor nanocrystals in the filtrate without the needfor washes or sample removal. Analyte concentration, in virtually anycontext where a specific binder exists for that analyte, can bedetermined. Thus, almost any analyte, chemical or biological, organic orinorganic may be detected in this manner.

The detection is achieved by competition between analyte in the sampleand analyte conjugated to semiconductor nanocrystals or conjugated to asemiconductor nanocrystal-encoded microsphere which is small enough topass through the filter pores. Separation is achieved because thereaction is free to pass through a microporous filter in which the poresare a smaller size than the diameter of the microsphere. Thus themicrosphere cannot pass through the filter (see FIG. 5). Semiconductornanocrystal-conjugates or semiconductor nanocrystal encoded microspheresare of small enough size to pass through the filter and will do sounless bound to the microspheres. If a known amount of semiconductornanocrystals are applied to the upper level, with the analyte sample,allowed to bind for a predetermined time and then passed through thefilter, the concentration of the analyte is determined by measuring thelevel of fluorescence present in the lower level. Fluorescence in theupper level is not detected either by removal of the upper level orbecause the membrane is opaque to the excitation source.

Detection is carried out by a spectral sensing device and separation ofdifferent semiconductor nanocrystal spectra is achieved by the use ofband-pass filters or by measuring light emission across the spectrum.Detection can be achieved by a static detection device or by a scanningdetector, as described above.

Semiconductor Nanocrystals as Detection Reagents in Probe-Based Assays

Semiconductor nanocrystals can also be used as sensitive detectionagents in probe-based assays for the detection of target nucleic acidsequences in test samples. Probes for use in these assays are designedfrom either conserved or nonconserved regions of the targetpolynucleotide of interest, using techniques well known in the art.Generally, probes are developed from nonconserved or unique regions whenmaximum specificity is desired, and from conserved regions when assayingfor regions that are closely related to, for example, different membersof a multigene family or in related species.

Polymerase chain reaction (PCR) is a technique for amplifying a desiredtarget nucleic acid sequence contained in a nucleic acid molecule ormixture of molecules. In PCR, a pair of primers is employed in excess tohybridize to the complementary strands of the target nucleic acid. Theprimers are each extended by a polymerase using the target nucleic acidas a template. The extension products become target sequences themselvesafter dissociation from the original target strand. New primers are thenhybridized and extended by a polymerase, and the cycle is repeated togeometrically increase the number of target sequence molecules. PCR isdescribed in U.S. Pat. Nos. 4,683,195 and 4,683,202, which areincorporated herein by reference in their entireties.

The Ligase Chain Reaction (LCR) is an alternate method for nucleic acidamplification. In LCR, probe pairs are used which include two primary(first and second) and two secondary (third and fourth) probes, all ofwhich are used in molar excess to target. The first probe hybridizes toa first segment of the target strand, and the second probe hybridizes toa second segment of the target strand, the first and second segmentsbeing contiguous so that the primary probes abut one another in 5′phosphate-3′ hydroxyl relationship. Thus, a ligase can covalently fuseor ligate the two probes into a fused product. In addition, a third(secondary) probe can hybridize to a portion of the first probe and afourth (secondary) probe can hybridize to a portion of the second probein a similar abutting fashion. If the target is initiallydouble-stranded, the secondary probes will also hybridize to the targetcomplement in the first instance. Once the ligated strand of primaryprobes is separated from the target strand, it will hybridize with thethird and fourth probes which can be ligated to form a complementary,secondary ligated product. By repeated cycles of hybridization andligation, amplification of the target sequence is achieved. Thistechnique is described in, e.g., European Publication No. 320,308,published Jun. 16, 1989 and European Publication No. 439,182, publishedJul. 31, 1991

More particularly, in the above methods, once the primers or probes havebeen sufficiently extended and/or ligated, they are separated from thetarget sequence, for example, by heating the reaction mixture to a “melttemperature” which dissociates the complementary nucleic acid strands.Thus, a sequence complementary to the target sequence is formed. A newamplification cycle can then take place to further amplify the number oftarget sequences by separating any double-stranded sequences, allowingprimers or probes to hybridize to their respective targets, extendingand/or ligating the hybridized primers or probes and reseparating. Thecomplementary sequences that are generated by amplification cycles canserve as templates for primer extension or fill the gap of two probes tofurther amplify the number of target sequences. Typically, a reactionmixture is cycled between 20 and 100 times, more typically between 25and 50 times. In this manner, multiple copies of the target sequence andits complementary sequence are produced. Thus, primers initiateamplification of the target sequence when it is present underamplification conditions.

mRNAs may be amplified by reverse transcribing the mRNA into cDNA, andthen performing PCR (RT-PCR), as described above. Alternatively, asingle enzyme may be used for both steps as described in U.S. Pat. No.5,322,770, which is incorporated herein by reference. mRNA may also bereverse transcribed into cDNA, followed by asymmetric gap ligase chainreaction (RT-AGLCR) as described by Marshall et al. (1994) PCR Meth.App. 4:80-84.

Other known amplification methods which can be utilized in probe-basedassays include, but are not limited to, the “NASBA” or “3SR” (Guatelliet al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878 and Compton, J.Nature 350:91-92); Q-beta amplification; strand displacementamplification (Walker et al. Clin. Chem. 42:9-13 and European PatentApplication No. 684,315); and target mediated amplification(International Publication No. WO 93/22461).

Detection, both amplified and nonamplified, may be performed using avariety of heterogeneous and homogeneous detection formats. Examples ofheterogeneous detection formats are disclosed in Snitman et al., U.S.Pat. No. 5,273,882; Urdea et al., U.S. Pat. No. 5,124,246; Ullman et al.U.S. Pat. No. 5,185,243; and Kourilsky et al., U.S. Pat. No. 4,581,333,all of which are incorporated herein by reference in their entireties.Examples of homogeneous detection formats are described in Caskey etal., U.S. Pat. No. 5,582,989; and Gelfand et al., U.S. Pat. No.5,210,015, which are incorporated herein by reference in theirentireties. Also contemplated and within the scope of the presentinvention is the use of multiple probes in hybridization assays, toimprove sensitivity and amplification of the target signal. See, forexample, Caskey et al., U.S. Pat. No. 5,582,989; and Gelfand et al.,U.S. Pat. No. 5,210,015; which are incorporated herein by reference intheir entireties.

Thus, in one embodiment, the present invention generally comprises thesteps of contacting a test sample suspected of containing a targetpolynucleotide sequence with amplification reaction reagents comprisingan amplification primer, and a detection probe that can hybridize withan internal region of the amplicon sequences. Probes and primersemployed according to the method provided herein are labeled withcapture and detection labels, wherein probes are labeled with one typeof label and primers are labeled with another type of label.Additionally, the primers and probes are selected such that the probesequence has a lower melt temperature than the primer sequences. Theamplification reagents, detection reagents and test sample are placedunder amplification conditions whereby, in the presence of targetsequence, copies of the target sequence (an amplicon) are produced. Inthe usual case, the amplicon is double-stranded because primers areprovided to amplify a target sequence and its complementary strand. Thedouble-stranded amplicon then is thermally denatured to producesingle-stranded amplicon members. Upon formation of the single-strandedamplicon members, the mixture is cooled to allow the formation ofcomplexes between the probes and single-stranded amplicon members.

As the single-stranded amplicon sequences and probe sequences arecooled, the probe sequences preferentially bind the single-strandedamplicon members. After the probe/single-stranded amplicon memberhybrids are formed, they are detected. Standard heterogeneous assayformats are suitable for detecting the hybrids using the detectionlabels and capture labels present on the primers and probes. The hybridscan be bound to a solid phase reagent by virtue of the capture label anddetected by virtue of the detection label. Either the capture label ordetection label comprises semiconductor nanocrystals. In cases where thedetection label is directly detectable, the presence of the hybrids onthe solid phase can be detected using techniques described above. Incases where the label is not directly detectable, the captured hybridscan be contacted with a conjugate, which generally comprises a bindingmember attached to a semiconductor nanocrystal label. The conjugatebecomes bound to the complexes and the presence of the conjugate on thecomplexes can be detected with the directly detectable semiconductornanocrystal label. Thus, the presence of the hybrids on the solid-phasereagent can be determined. Wash steps are typically employed during theabove reactions to wash away unhybridized amplicon or probe as well asunbound conjugate.

The heterogeneous assays can be conveniently performed using a solidphase support that carries an array of nucleic acid molecules. Sucharrays are useful for high-throughput and/or multiplexed assay formats,described in more detail below. Various methods for forming such arraysfrom pre-formed nucleic acid molecules, or methods for generating thearray using in situ synthesis techniques, are generally known in theart. (See, for example, Dattagupta, et al., European Publication No.234,726; U.S. Pat. No. 5,700,637 to Souther; U.S. Pat. No. 5,143,854 toPirrung, et al.; International Publication No. WO 92/10092; and, Fodor,et al. (1991) Science 251:767-777.

Although the target sequence above is described as single-stranded, thetarget may also be double-stranded and separated from its complementprior to hybridization with the amplification primer sequences. Further,while the amplification primers initiate amplification of the targetsequence, the detection (or hybridization) probe is not involved inamplification. Detection probes are generally nucleic acid sequences oruncharged nucleic acid analogs such as, for example, peptide nucleicacids which are disclosed in International Publication No. WO 92/20702;morpholino analogs which are described in U.S. Pat. Nos. 5,185,444,5,034,506 and 5,142,047; and the like. Depending upon the type of labelcarried by the probe, the probe is employed to capture or detect theamplicon generated by the amplification reaction.

While the length of the primers and probes can vary, the probe sequencesare selected such that they have a lower melt temperature than theprimer sequences. Hence, the primer sequences are generally longer thanthe probe sequences. Typically, the primer sequences are in the range ofbetween 20 and 50 nucleotides long, more typically in the range ofbetween 20 and 30 nucleotides long. The typical probe is in the range ofbetween 10 and 25 nucleotides long.

As explained above, aptamers are single- or double-stranded DNA orsingle-stranded RNA molecules that recognize and bind to a desiredtarget molecule by virtue of their shapes. See, e.g., PCT PublicationNos. WO92/14843, WO91/19813, and WO92/05285. The SELEX procedure,described in U.S. Pat. No. 5,270,163 to Gold et al., Tuerk et al. (1990)Science 249:505-510, Szostak et al. (1990) Nature 346:818-822 and Joyce(1989) Gene 82:83-87, can be used to select for RNA or DNA aptamers thatare target-specific. In the SELEX procedure, an oligonucleotide isconstructed wherein an n-mer, preferably a randomized sequence ofnucleotides thereby forming a “randomer pool” of oligonucleotides, isflanked by two polymerase chain reaction (PCR) primers. The construct isthen contacted with a target molecule under conditions which favorbinding of the oligonucleotides to the target molecule. Thoseoligonucleotides which bind the target molecule are: (a) separated fromthose oligonucleotides which do not bind the target molecule usingconventional methods such as filtration, centrifugation, chromatography,or the like; (b) dissociated from the target molecule; and (c) amplifiedusing conventional PCR technology to form a ligand-enriched pool ofoligonucleotides. Further rounds of binding, separation, dissociationand amplification are performed until an aptamer with the desiredbinding affinity, specificity or both is achieved. The final aptamersequence identified can then be prepared chemically or by in vitrotranscription. Semiconductor nanocrystals are readily used in thedetection of such aptamers.

Specific embodiments of the above-described probe-based assays aredescribed in further detail below.

Semiconductor Nanocrystals for Detection Reagents in Fluorescence InSitu Hybridization

In another embodiment of the invention, FISH assays using asemiconductor nanocrystal as a detectable label are disclosed.Techniques for performing various types of FISH assays are well known inthe art and described in, e.g., Raap, A. K. (1998) Mutation Res.400:287-298; Speel et al. (1998) Histochem. Cell. Biol. 110:571-577;Nath and Johnson (1997) Biotech. Histochem. 73:6-22; Swiger and Tucker(1996) Environ. Molec. Mutagen. 27:245-254; Kitadai et al. (1995) Clin.Cancer Res. 1:1095-1102; Heiskanen et al. (1995) Genomics 30:31-36; andHeiskanen et al. (1994) BioTechniques 17:928-933. Semiconductornanocrystals can be substituted for the fluorescent labels normally usedin each of these techniques. The advantages of nucleic acid probeslabeled with semiconductor nanocrystals is that multiple probes directedat distinct target oligonucleotides can be used simultaneously by virtueof the fact that a plurality of populations of semiconductornanocrystals can be made with nonoverlapping emission spectra, each ofwhich can be excited with a single source and wavelength of light. Theability to “multiplex” assays in this manner is especially useful whenthe specimen to be analyzed contains a limited source of cells ortissues, e.g., rare cells, fetal cells in maternal blood, cancer cellsin blood or urine samples, blastomeres, or the like. By multiplexing,multiparametric information at the single cell level may be collected.See, e.g., Patterson et al. (1998) Cytometry 31:265-274; Borzi et al.(1996) J. Immunol. Meth. 193:167-176; Wachtel et al. (1998) Prenat.Diagn. 18:455-463; Bianchi (1998) J. Perinat. Med. 26:175-185; and Munne(1998) Mol. Hum. Reprod. 4:863-870.

Semiconductor nanocrystals of many colors can be chemically linked tonucleic acid (DNA or RNA) or indirectly linked to streptavidin/biotinthat binds to nucleic acid. Semiconductor nanocrystals bind to DNAprimers or incorporate into nucleic acid by using semiconductornanocrystal-linked nucleotide(s). PCR can be used to generate nucleicacid fragments for FISH probes. Semiconductor nanocrystals can also bechemically attached to a nucleic acid containing the sequence ofinterest. Alternatively, biotin molecules can be attached tooligonucleotide primers, or incorporated into nucleic acid of interestby using biotinlyated nucleotides in PCR. Semiconductor nanocrystalsattached to streptavidin will then be linked to biotin in the nucleicacid probe. These semiconductor nanocrystal-FISH probes can be use forin situ hybridization for DNA (see Example 5; see also Dewald et al.(1993) Bone Marrow Transplantation 12:149-154; Ward et al. (1993) Am. J.Hum. Genet. 52:854-865; Jalal et al. (1998) Mayo Clin. Proc. 73:132-137;Zahed et al. (1992) Prenat. Diagn. 12:483-493; Neuhaus et al. (1999)Human Pathol. 30:81-86; Buno et al. (1998) Blood 92:2315-2321; Munne(1998) Mol. Hum. Reprod. 4:863-870, and RNA (see Example 6; Kitadai etal. (1995) Clin. Cancer Res. 1:1095-1102). The results can be analyzed,for example, under an epi fluorescence microscope. Semiconductornanocrystal-FISH probe or probes for DNA and RNA together (see Example7; Wachtel et al. (1998) Prenat. Diagn. 18:455-463), or for RNA andsurface immunophenotyping together (see Example 8; Patterson et al.(1998) Cytometry 31:265-274; Borzi et al. (1996) J. Immunol. Meth.193:167-176) can be used to identify, sort, and analyze rare cellssimultaneously. In the case where only short oligonucleotidesemiconductor nanocrystals-FISH probes (forward and reverse primers) forRNA or DNA are available, sensitivity of probes can be increased throughPCR/FISH or RT-PCR/FISH. This can be accomplished by incorporating asemiconductor nanocrystal-dNTP into the in situ PCR or RT-PCR reaction.(see Example 9; Patterson et al. (1993) Science 260:976-979; Pattersonet al. (1998) Cytometry 31:265-274). The detection system may be amicroscope, or flow cytometer, or detector capable of measuring thewavelength of light emitted from the different semiconductornanocrystals.

FISH technologies are widely used in research and clinical molecularcytogenetics, pathology and immunology laboratories. Semiconductornanocrystal-DNA probes can be use to detect amplification (e.g.,HER2/neu, c myc genes amplification), addition (e.g., trisomy 21, 13,18), deletion (e.g., 45×, Turner's Syndrome), translocation (e.g.,BCR/ABL in CML) of DNA in the nuclei.

Semiconductor nanocrystal-RNA probes can be used to localize and tomonitor expression of genes (mRNA) in the cell. This is especiallyuseful for detecting rare cells (e.g. fetal cells in maternal blood,cancer cells for monitoring disease recurrence).

In the case where only forward and reverse primers (with or withoutsemiconductor nanocrystals attached) are available, PCR/FISH orRT-PCR/FISH can be used to amplify the target DNA or RNA to increasesensitivity. This can be accomplished by incorporating a semiconductornanocrystal-dNTP into the in situ PCR or RT-PCR reaction.

Semiconductor nanocrystals can be conjugated to antibodies, to a proteinof interest (antigen) to detect protein expression and/or sort out cellsof interest. Multiple semiconductor nanocrystals-antibody(ies),semiconductor nanocrystals-nucleic acid probes for RNA and or DNA can beused to hybridize with cells in the same or sequential reaction. Cellsof interest in the population (rare cells) can then be identified andanalyzed for DNA (for genetic composition) or mRNA (for gene expression)simultaneously.

Specimens for FISH Assays: Specimens can be cells (alive or fixed) ornuclei in suspension or attached to microscope slides or other solidsupports or paraffin embedded tissue sections containing one, or morethan one specimen, or frozen tissue sections or fine needle aspirate.FISH can be performed on metaphase or interphase cells or directly ontoDNA strands.

The specimen for the FISH assay is prepared using well known methodsdepending on the specimen type, for example: peripheral blood (Hack etal., eds., (1980), supra; Buno et al. (1998), supra; Patterson et al.(1993), supra; Patterson et al. (1998), supra; Borzi et al. (1996),supra); bone marrow (Dewald et al. (1993), supra; Hack et al., eds.,(1980), supra); amniocytes (Ward et al. (1993), supra; Jalal et al.(1998), supra); CVS (Zahed et al. (1992), supra); paraffin embeddedtissue sections (Kitadai et al. (1995); supra; Neuhaus et al. (1999),supra); fetal cells (Wachtel et al. (1998), supra; Bianchi (1998),supra); and blastomeres (Munne (1998), supra).

Optimization of Conditions for FISH Assays: The conditions of the FISHassays exemplified below can be optimized depending on the nature ofdifferent probes (e.g., whether the nucleic acid sequence is highlyrepetitive or not) or mixtures of probes. The assays are carried out atroom temperature to 100° C. but are typically carried out in the rangeof 37° C. to 80° C.

Semiconductor Nanocrystals as Detection Reagents in Signal AmplificationAssays

In yet another embodiment of the invention, a method is disclosed forusing semiconductor nanocrystals as a signal-generating label andsemiconductor nanocrystal conjugates as the detection reagent in signalamplification assay formats. This type of signal amplification providesseveral advantages over currently employed methods for detecting thesignal in signal-amplification assays. Among these advantages is theability to detect multiple analytes in the same sample simultaneouslywith high sensitivity.

Semiconductor nanocrystals of one or more colors are individuallyconjugated to distinct molecules (a “semiconductornanocrystal-conjugate”) that specifically recognize an amplificationcomplex generated in response to the presence of an analyte in a sample.A semiconductor nanocrystal-conjugate can be, for example, the labelin 1) a DNA hybridization assay, (see Example 10), or 2) abiotin/avidin-layered amplification assay (see Example 11). Thedetection system is a device capable of measuring and distinguishing thewavelength of light emitted from semiconductor nanocrystals of one ormore colors.

Semiconductor Nanocrystals for Use in Multiplexed, Single Tube Assays

In still another embodiment of the invention, an HTS assay usingsemiconductor nanocrystals as multiplexed detection reagents isprovided. Semiconductor nanocrystals of a particular color areconjugated by one of the techniques described in Bruchez et al. supra,Bruchez, supra, Chan et al., supra, or by any technique known in the artfor attaching or conjugating proteins, nucleic acids, and the like. See,e.g., Hermanson (1996) Bioconjugate Techniques (Academic Press).

The HTS assay is performed in the presence of various concentrations ofa candidate compound. The semiconductor nanocrystal emission ismonitored as an indication of the effect of the candidate compound onthe assay system. This technique is amenable to any of the conventionaltechniques with the exception of chemiluminescence. For example,fluorescence reading using a semiconductor nanocrystal-conjugated ligandor receptor to monitor binding thereof to a bead-bound receptor orligand, respectively, may be used as a flexible format to measure thesemiconductor nanocrystal emission associated with the beads. Themeasure of semiconductor nanocrystal emission associated with the beadscan be a function of the concentration of candidate compound and, thus,of the effect of the candidate compound on the system. In addition,semiconductor nanocrystals can be used as a multicolor scintillant todetect the binding of a radiolabeled ligand or receptor with asemiconductor nanocrystal-conjugated receptor or ligand, respectively. Adecrease in scintillation would be one result of inhibition by thecandidate compound of the ligand-receptor pair binding.

Semiconductor Nanocrystals for Use in High-Throughput Sequence Analyses

Semiconductor nanocrystals conjugated to nucleic acids may be used inhigh-throughput DNA sequencing and DNA fragment analysis. To describethese sequencing reactions briefly, four reactions are performed todetermine the positions of the four nucleotide bases within a DNAsequence. Using a DNA sample as a template, a chain of DNA issynthesized from a pool of nucleotides containing the fourdeoxynucleotides and one additional dideoxynucleotide. For example, inthe adenine sequencing reaction, DNA is synthesized from a mixture thatincludes all four deoxynucleotides (dATP, dGTP, dCTP, dTTP) plusdideoxyadenosine triphosphate (ddATP). The enzyme DNA polymerase willsynthesize the new chain of DNA by linking dNTPs. Occasionally DNApolymerase will incorporate a ddATP instead of a dATP. The ddATP in thenascent chain will then terminate the synthesis of that chain of DNA dueto the lack of the 3′ hydroxyl group as a connection to the next dNTP.Thus the DNA products from the adenine sequencing reaction will be aheterogeneous mixture of DNA that vary in length with each chainterminated at a position corresponding to adenine.

The four DNA sequencing reactions are resolved by size by polyacrylamidegel electrophoresis. With singly radiolabeled (³²P or ³⁵S) DNA, the fourreactions are loaded into four individual lanes. The resolved productsof differing sizes result in a pattern of bands that indicate theidentity of a base at each nucleotide position. This pattern across thefour lanes can be read like a simple code corresponding to thenucleotide base sequence of the DNA template. With fluorescentdideoxynucleotides, samples containing all four dideoxynucleotidechain-terminating reactions can be loaded into a single lane. Resolutionof the four dideoxynucleotide reactions is possible because of thedifferent fluorescent labels for each sample. For example, ddATP can beconjugated with a green fluorescent tag. The other three ddNTP(dideoxynucleotide triphosphate) are tagged with three differentfluorescent colors. Thus, each chain-terminating ddNTP is coded with adifferent color. When all four reactions are resolved in one lane on aDNA sequencing gel, the result is one ladder of bands having fourdifferent colors. Each fluorescent color corresponds to the identity ofthe nucleotide base and can be easily analyzed by automated systems.

However as previously discussed, multiple light sources are needed forexcitation of the four different fluorescent organic dye markers. Theuse of semiconductor nanocrystals as the fluorescent tags for eachdideoxynucleotide chain-terminating reaction simplifies the automationof high-throughput DNA sequencing since only a single light source isneeded to excite all four fluorescent tags. In addition, multiplexingwith semiconductor nanocrystals permits multiple sequencing reactions tobe conducted and analyzed simultaneously, thereby further increasing thethroughput of the assay.

In PCR (polymerase chain reaction)-based DNA typing and identification,short tandem repeat (STR) loci in the human genome are amplified by PCRusing primers that are labeled with fluorescent tags. The size of theseloci can differ or can coincide from person to person, or fromindividual subject to individual subject, and depends on geneticdifferences in the population. Usually multiple loci are examined. Anylocus that shows a size difference with another sample conclusivelyindicates that the two samples are derived from two differentindividuals. However, demonstrating that two samples originate from thesame individual is less conclusive. Unlike fingerprint patterns, thesize of STR loci can coincide between two individuals. However, thestatistical probability of multiple loci coinciding in size between twoindividuals decreases as the number of loci examined is increased. Usingconventional organic fluorescent dyes, a limitation to the number ofsamples resolved in a single lane (and thus high-throughput) is thenumber of the fluorescent tags available and the resolution of theemission spectra. Increasing the resolution of the fluorescent tags thuswould increase the capacity of the number of loci tested per lane on agel.

Semiconductor Nanocrystals as Detection Reagents in DNA-Based Assays

In still further embodiments of the invention, semiconductornanocrystals can be used in other hybridization formats, sequencespecific extension and oligo ligation assays, for detecting thepresence/absence of specific DNA target sequences.

DNA Hybridization: The single strands within DNA helices are heldtogether by virtue of the hydrogen bonds between complementary bases ineach strand. When double-stranded DNA (“dsDNA”) is subjected tophysical/chemical conditions which disrupt hydrogen bonds (i.e.,denature the DNA), the strands separate into single-stranded (“ssDNA”)molecules. When a mixture of different dsDNA molecules is denatured andthen returned to conditions in which hydrogen bonds betweencomplementary bases can reform, many of the complementary ssDNA strandswill “find” each other and hybridize (reanneal) to form the originaldsDNA molecules. The process of nucleic acid hybridization serves as thebasis for countless diagnostic tests: e.g., Dot Blots, Southern Blots,Northern Blots, and FISH Analysis.

Denaturation: DNA duplexes are held together by hydrogen bonds betweencomplementary base pairs as well as by hydrophobic interactions betweenthe stacked bases. These associations can be disrupted (i.e., DNA can bedenatured, or “melted”) by raising the temperature, decreasing the saltconcentration, increasing the alkalinity, or by adding various organicreagents. Since G-C base pairs form three hydrogen bonds, whereas A-Tbase pairs form two hydrogen bonds, A/T-rich DNA helices melt (denature)at lower temperatures and/or higher salt concentrations than G/C-richsequences. The amount of dsDNA that has become denatured(single-stranded, ssDNA) can be determined by a variety of physical andchemical means. The temperature at which 50% of the total base pairswithin the dsDNA molecules in a solution have become disrupted is calledthe melting temperature (Tm).

Renaturation (“reannealing,” “hybridization”): The stability of nucleicacid hybrids is dependent on environmental conditions; two of the mostimportant of which are temperature and salt concentration. In general,DNA duplexes are more stable at lower temperatures and higher saltconcentrations. When denatured DNA is cooled or subjected to higher saltconcentrations, it will spontaneously renature. Under ideal renaturationconditions and given sufficient time, mixtures of single-strandedfragments of DNA will reassociate (hybridize) with their complementarysequences. This property of nucleic acids is the basis upon which theassays disclosed herein are founded.

Stringency of Hybridization: When ssDNA molecules form hybrids, the basesequence complementarity of the two strands does not have to be perfect.Poorly matched hybrids (i.e. hybrids in which only some of thenucleotides in each strand are aligned with their complementary bases soas to be able to form hydrogen bonds) can form at low temperatures, butas the temperature is raised (or the salt concentration lowered) thecomplementary base-paired regions within the poorer hybrids dissociatedue to the fact that there is not enough total hydrogen bond formationwithin the entire duplex molecule to hold the two strands together underthe new environmental conditions. The temperature and/or saltconcentrations may be changed progressively so as to create conditionswhere an increasing percentage of complementary base pair matches isrequired in order for hybrid duplexes to remain intact. Eventually, aset of conditions will be reached at which only perfect hybrids canexist as duplexes. Above this stringency level, even perfectly matchedduplexes will dissociate. The stringency conditions for each uniquefragment of dsDNA in a mixture of DNA depends on its unique base paircomposition. The degree to which hybridization conditions requireperfect base pair complementarity for hybrid duplexes to persist isreferred to as the “stringency of hybridization.” Low stringencyconditions are those which permit the formation of duplex moleculeshaving some degree of mismatched bases. High stringency conditions arethose which permit only near-perfect base pair-matched duplex moleculesto persist. Manipulation of stringency conditions is key to theoptimization of sequence specific assays.

Nucleic acid probes: For each of the assay procedures described it isnecessary to have one or more nucleic acid fragments that have beenlabeled with semiconductor nanocrystals, so that when they bind to thetarget DNA sequence their presence can be easily detected. Probes canconsist of many nucleic acid types, depending on subtle aspects of thedetection method and sample to be detected. They may be single-strandedor denatured double-stranded DNA or RNA. Probes of dozens to severalhundred bases long can be artificially synthesized using oligonucleotidesynthesizing machines, or they may be derived from various types of DNAcloning. The critical aspects are that the probe must contain a nucleicacid strand that is at least partially complementary to the targetsequence to be detected, and the probe must be labeled with thesemiconductor nanocrystals of the invention so that its presence can bevisualized.

Semiconductor Nanocrystals for Use in Spectral Karyotyping

Spectral karyotyping is a FISH technique used on metaphase preparationsof chromosomes. See, e.g., Schrock et al. (1996) Science 273:494-497;and Macville et al. (1997) Histochem. Cell. Biol. 108:299-305. Inspectral karyotyping and fluorescence in-situ hybridization (FISH)measurements, chromosomes are stained using combinations of dyes atdifferent wavelengths and ratios. This acts to “barcode” differentchromosomes for detection. For example, chromosome analysis is typicallydone by staining (e.g., Giesma stain). In a similar way, semiconductornanocrystals can be used to barcode chromosomes using the techniquesdescribed above. In particular, as explained above, an arbitrary numberof codes can be generated without the need to control the intensity ofany spectral peak within the code. In this case, DNA probes are labeledwith semiconductor nanocrystals that emit different colors to “paint”different chromosomes. Since the chromosomes are spatially separatedduring spectral karyotyping measurements, a barcoding system can be usedto label each chromosome. The chromosomes act as the solid support forthe coding semiconductor nanocrystal. By using this type of codingsystem, it is possible to generate an arbitrary number (more than 48) ofunambiguous codes for spectral karyotyping.

In addition to barcoding entire chromosomes, it is also possible tobarcode sections of a chromosome. In this way, this technique canreplace traditional chromosome staining.

Normally stains are used to label different spatially distinct regionsof a chromosome. Different colors are used to stain different regions.With semiconductor nanocrystals, different sections on each chromosomecan be painted with different barcodes. In this case, the spatiallocations on the chromosome act as the substrate for different codes.This allows a very large number of regions to be labeled simultaneously.

Semiconductor nanocrystal-labeled probes for chromosome painting can begenerated using techniques well known in the art, such as, but notlimited to, micro-dissected PCR DNA or cloned DNAs (e.g. from YACS,BACS, PACs etc.).

III. EXPERIMENTAL

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

Reagents for use in the following examples may be purchased fromcommercial sources, and used according to the manufacturers' directions.In the cloning of DNA fragments, except where noted, all DNAmanipulations are done according to standard procedures. See, e.g.,Sambrook et al., supra. Restriction enzymes, T4 DNA ligase, E. coli, DNApolymerase I, Klenow fragment, and other biological reagents arepurchased from commercial suppliers and used according to themanufacturers' directions.

For the following examples, water-soluble core-shell nanocrystals aresynthesized as described in Bruchez et al. (1998) Science 281:2013-2016.Affinity molecules are linked directly or indirectly to a semiconductornanocrystal using chemical techniques well known in the art. See, e.g.,U.S. Pat. No. 5,990,479 to Alivisatos et al; Bruchez et al. (1998)supra; and Chan et al. (1998) Science 281:2016-2018; Haugland, supra;and Hermanson “Bioconjugate Techniques” (Academic Press, NY).

Example 1 Sandwich QISA

This example describes a sandwich QISA in which an analyte-specificantibody, or a plurality of different analyte-specific antibodies eachof which is capable of specifically recognizing and binding to adistinct analyte, are immobilized on a solid support and analytes boundthereto are detected using one or a plurality of semiconductornanocrystal/analyte-specific antibody conjugates.

A. A primary antibody (species 1 as illustrated in FIG. 1A) specific forthe analyte to be detected in the sample is immobilized on a solidsupport, e.g., a well of a 96-well plate, using well-establishedtechniques. The concentration of support-bound primary antibody isselected to be appropriate for the assay (1 ng/ml to 10 mg/ml).Optionally, the support is washed to remove unbound antibody.

Any portion of the solid support to which no antibody is bound istreated with an agent to reduce nonspecific binding thereto, e.g., 1%BSA+1% antibody species 2 antiserum (e.g., an antibody that is distinctfrom the first capturing antibody species) in phosphate-buffered saline(PBS). Optionally, the support is washed to remove excess blockingagent.

A sample containing or suspected of containing the analyte of interestis added to the treated solid support and incubated for a time (30seconds to 48 hours) sufficient to bind the analyte to the primaryantibody. Optionally, the support is again washed to remove nonspecificand unbound analyte molecules in the sample.

A secondary antibody (species 3 distinct from the primary antibody)conjugated to a single population of the same composition ofsemiconductor nanocrystals is added at a known concentration (1 ng/ml to10 mg/ml) and incubated for a time (30 seconds to 48 hours) sufficientto bind the secondary antibody to the support-bound analyte. Optionally,the support is washed to remove unbound semiconductornanocrystal-conjugated secondary antibody.

The results of assay are determined by measuring the intensity of lightemitted from the semiconductor nanocrystals specifically bound to thesolid support at wavelengths corresponding to the semiconductornanocrystals component of the conjugate.

In particular, a QISA was performed by coating the wells of a 96-wellplate with 10 μg/ml of rabbit IgG in PBS. After coating, the plate waswashed and unbound sites were blocked with BSA. The plates were blockedfor one hour at room temperature prior to washing and concentrations ofbiotinylated goat anti-rabbit IgG ranging from 0.1 nM to 100 nM (in PBS)were added. This was incubated in the wells for one hour at roomtemperature and then washed. 100 nM nanocrystal conjugates (streptavidinconjugates) were added which had an emission peak at 580 nm. Thenanocrystal conjugates were incubated in the wells for one hour at roomtemperature and then washed. The plate was read on a Molecular Dynamicsspectramax Gemini plate reader.

Results are shown in FIG. 2. As can be seen, fluorescent intensityincreased with the concentration of biotinylated antibody used,demonstrating the usefulness of semiconductor nanocrystals in thiscontext.

B. A sample can be analyzed simultaneously for the presence of more thanone analyte in a single assay using a sandwich QISA as disclosed herein.A species of primary antibody capable of recognizing and specificallybinding to one of each of the analytes of interest is bound to the solidsupport, the support is, optionally, washed, blocked and washed again,as described above. The sample is then added to the support andincubated for a time sufficient for each analyte to be recognized by andbind to its corresponding primary antibody. The support is, optionally,washed to remove unbound sample. A plurality of distinct secondaryantibodies, each of which is capable of recognizing and binding to oneof each of the analytes of interest and is conjugated to a distinctspecies of a semiconductor nanocrystal. Each distinct species of asemiconductor nanocrystal has distinctly detectable emission properties

The support-bound semiconductor nanocrystal-antibody conjugates aresimultaneously irradiated with blue light and the plurality of emissionwavelengths corresponding to each species of semiconductor nanocrystalis monitored using an appropriate apparatus.

Example 2 Direct Capture QISA

This example describes a direct capture QISA in which a samplecontaining or suspected of containing an analyte of interest or aplurality of different analytes of interest, is adsorbed onto a solidsupport and adsorbed analytes are detected using one or a plurality ofsemiconductor nanocrystal/analyte-specific conjugates, respectively.

A sample is adsorbed onto a solid support using well-establishedtechniques for a time sufficient to effect adsorption (30 seconds to 48hours). Optionally, the support is washed to remove unbound molecules.Nonspecific binding is minimized by treating the solid support to whichsample has been adsorbed with an appropriate blocking agent, e.g., 1%BSA+1% serum (same species as primary antibody) in PBS. The support isagain, optionally, washed to remove excess blocking agent,

For detecting a single analyte, analyte-specific antibody conjugated toa single population of the same composition of semiconductornanocrystals is added at a known concentration (1 ng/ml to 10 mg/ml) andincubated with the sample-adsorbed solid support for a time sufficientfor the antibody to recognize and bind to its corresponding antibody (30seconds to 48 hours). The support is, optionally, washed to removeunbound semiconductor nanocrystal-conjugated antibody.

Results of the assay are determined by measuring the intensity of lightemitted at a wavelength that correspond to the semiconductor nanocrystalin the conjugate.

In a manner similar to that described in Example 1B, the direct captureQISA can be adapted to detect multiple analytes in a single sample byusing a plurality of primary antibody species, each of which isconjugated to a semiconductor nanocrystal with distinctly detectableemission properties. The support-bound semiconductornanocrystal-antibody conjugates are simultaneously irradiated with bluelight and the plurality of emission wavelengths corresponding to eachsemiconductor nanocrystal is detected using an appropriate apparatus.

Example 3 Fluid-Phase QISA

This example describes a fluid-phase QISA in which a fluid-phase samplecontaining or suspected of containing an analyte of interest or aplurality of different analytes of interest, is adsorbed onto asolid-phase substrate and adsorbed analytes are detected using one or aplurality of semiconductor nanocrystal/analyte-specific antibodyconjugates.

A sample containing or suspected of containing analyte is adsorbed onto,for example, latex microspheres (see, e.g., Bangs Laboratories, Inc.,TechNote#25) by incubating the microspheres with the sample for anappropriate amount of time (30 seconds to 48 hours). Optionally, themicrospheres are washed, e.g., by centrifugation, to remove unboundsample. Microspheres to which analyte have been adsorbed are incubatedwith semiconductor nanocrystal-conjugated analyte-specific antibody (1ng/ml to 10 mg/ml) for a period of time sufficient to allow binding ofthe antibody to the adsorbed analyte (30 seconds 48 hours). Optionally,the microspheres are washed to remove unbound semiconductornanocrystal-conjugated antibodies.

The amount of semiconductor nanocrystal fluorescence associated with themicrospheres can be detected using flow cytometry or a staticfluorimeter capable of distinguishing the different spectrum ofemissions from the semiconductor nanocrystal conjugates.

In a manner similar to that described in Example 1B, the fluid-phaseQISA can be adapted to detect multiple analytes in a single sample byusing a plurality of primary antibody species, each of which isconjugated to a semiconductor nanocrystal with distinctly detectableemission properties. The support-bound semiconductornanocrystal-antibody conjugates are irradiated with blue light and theplurality of emission wavelengths corresponding to each semiconductornanocrystal is detected using flow cytometry or a static fluorimeter asdescribed above.

Example 4 Fluid-Phase QISA

This example describes a fluid-phase QISA in which a fluid-phase samplecontaining or suspected of containing an analyte of interest or aplurality of different analytes of interest, is incubated with and boundto a primary antibody, or a plurality of primary antibodies capable ofspecifically recognizing and binding to an analyte. The primary antibodyis immobilized on a microsphere. Bound analyte or analytes are detectedusing one or a plurality of semiconductor nanocrystal/analyte-specificantibody conjugates.

A primary polyclonal or monoclonal antibody (species 1) specific for theanalyte to be detected in the sample is immobilized onto microspheresusing well-established techniques. The concentration of primary antibodyshould be appropriate for the assay (1 ng/ml to 10 mg/ml), as describedin Example 1. Optionally, the microspheres are washed to remove unboundprimary antibody.

A sample containing or suspected of containing an analyte is incubatedwith microspheres to which have been conjugated an analyte-specificantibody for a period of time (30 seconds to 48 hours) sufficient toeffect binding of an analyte to its respective antibody. Optionally, themicrospheres are washed to remove unbound sample and/or analyte.

Primary antibody-microsphere conjugates which have been incubated withthe sample are then incubated with a semiconductornanocrystal/analyte-specific secondary antibody conjugates at aconcentration (1 ng/ml to 10 mg/ml) and for a period of time (30 secondsto 48 hours) sufficient to effect binding of the semiconductornanocrystal-conjugated to the microsphere-immobilized analyte.Optionally, the microspheres are washed to remove unbound semiconductornanocrystal-conjugated antibodies.

The amount of semiconductor nanocrystal fluorescence associated with themicrospheres can be detected using flow cytometry or a staticfluorimeter capable of distinguishing the different spectrum ofemissions from the semiconductor nanocrystal conjugates.

In a manner similar to that described in Example 3, the fluid-phase QISAcan be adapted to detect multiple analytes in a single sample by using aplurality of primary antibody species, each of which is specific for apreselected analyte, conjugated to a microphere, and a plurality ofsemiconductor nanocrystal/analyte-specific secondary antibodyconjugates, in which each conjugate contains a semiconductor nanocrystalhaving distinctly detectable emission properties. The support-boundsemiconductor nanocrystal-antibody conjugates are irradiated with bluelight and the plurality of emission wavelengths corresponding to eachsemiconductor nanocrystal is detected using flow cytometry or a staticfluorimeter as described above.

Example 5 DNA-Based FISH Assay

The specimen for FISH assay is prepared using various published methodsdepending on the specimen type, as described above.

A. Double-stranded specimen DNA and semiconductor nanocrystal-FISHprobe(s) can be denatured together or separately to theirsingle-stranded form. Denaturation of semiconductor nanocrystal-FISHprobes consisting of synthetic oligonucleotides without secondarystructure is not necessary. The specimen and probes in single-strandedform are allowed to hybridize to their complimentary sequence inhybridization buffer optionally containing blocking DNA (5 minutes toovernight at 25° C.-70° C.). The excess semiconductor nanocrystal-FISHprobes are removed using a series of stringent washes. (e.g., 0.4×SSC to2×SSC with or without formamide at 25° C.-72° C.). Optionally, thespecimen is air dried and counterstained to localize the nuclei.

More particularly, semiconductor nanocrystal-FISH probe(s) is added tothe hybridization mix containing 50% formamide, 10% dextran sulphate,1×SSC (saline citrate), 3 μg Cot1 DNA. The probe mixture is deposited onthe slide and a coverslip is placed on the probe. The slide is placed ona hot plate for 3 minutes at 72° C. to denature the specimen and probeDNA. The specimen and probes in single-stranded form are allowed tohybridize to their complimentary sequence for 5 minutes to overnight at25° C. to 45° C. The excess semiconductor nanocrystal-FISH probes areremoved by washing the slide in 2×SSC, 0.3% NP-40 for 2 minutes. Thespecimen is air dried and counterstained to localize the nuclei.

The fluorescence signals of the semiconductor nanocrystal-FISH probes isviewed under an epi-fluorescence microscope. Alternatively, the signalsare measured using a laser detector.

B. Whole Chromosome Paints:

Human chromosomes 1, 2, and 3 were each microdissected, PCR amplifiedand labeled with biotin-dUTP as described in Meltzer et al. (1992) Mat.Genet. 1:24-28. Slides with metaphase spreads from peripheral bloodlymphocytes were prepared using conventional cytogenetics techniques anddenatured for 2 minutes at 72° C. in 70% formamide, 2×SSC (salinecitrate). The slides were then dehydrated 1 minute each with a series of70%, 85% and 100% ethanol and air-dried. Hybridization mixture (10 ng/μlDNA of each whole chromosome probe in 50% formamide, 10% dextransulphate, 1×SSC, 3 μg Cot1 DNA) was denatured for 5 minutes at 72° C.and placed on the slides. A cover slip was placed on each slide andsealed with rubber cement. The slides were incubated at 37° C. overnightin a humidified chamber. After hybridization, the excess probes werewashed away three times with 2×SSC, 50% formamide at 42° C. for 3minutes each, followed by a rinse with 2×SSC at ambient temperature for3 minutes. The slides were then blocked with 4×SSC, 0.1% Tween 20, 1%BSA (Bovine Serum Albumin) for 30 minutes to prevent nonspecificbinding. Finally the slides were incubated with 40 nM of 630 nmsemiconductor nanocrystal-streptavidin in PBS (Phosphate bufferedsaline), 1% BSA, 10 mM MgCl₂ at ambient temperature for 1 hour. Theexcess semiconductor nanocrystal-streptavidin and salts were removed byrinsing the slides in PBS and in 10 mM Phosphate buffer. The slides werethen air dried and examined with a fluorescence microscope.

Chromosome pairs 1, 2 and 3 were readily visualized using thefluorescence microscope, demonstrating the utility of usingsemiconductor nanocrystals in this context.

C. Fiber FISH

Semiconductor nanocrystals were used in a Fiber FISH format essentiallyas described by Heiskanen et al. (1994) Biotechniques 17:928-933; andHeiskanen et al. (1995) Genomics 30:31-36. In particular, a 10 kb DNAprobe was labeled by nick-translation using biotin-11-dUTP (SigmaChemical). Fiber FISH slides were prepared as described in Heiskanen etal., supra. The slides were denatured in 70% formamide, 2×SSC at 72° C.for 4 minutes followed by dehydration with a series of 70%, 85% and 100%ethanol. Biotin labeled DNA probes 2.5-7.5 ng/μl were added tohybridization mix containing 50% formamide, 10% dextran sulfate, 2×SSC,0.2 μg/μl herring sperm DNA, 0.25 μg/μl Cot-1 DNA. The mixture wasplaced on slides, a coverslip was placed over each slide and hybridizedovernight at 37° C. in a humidified chamber. Excess probes were removedby washing the slides three times with 50% formamide, 2×SSC for 5minutes each, two times with 2×SSC for 5 minutes each and a final washin 0.5×SSC for 5 minute all at 45° C. Slides were incubated in 5% BSA,4×SSC, 0.05% Tween 20 for 15 minute at 37° C. to block nonspecificbinding. Biotinylated probes were detected by incubating in 40 nMsemiconductor nanocrystal-streptavidin in PBS, 1% BSA and 10 mM MgCl₂,followed by biotinylated anti avidin antibody and another layer ofsemiconductor nanocrystal-streptavidin conjugate. The slides were rinsedin PBS, stained with 5 ug/ml DAPI (Sigma Chemical) and examined in afluorescence microscope.

The DNA probes were readily visualized using a fluorescence microscope,demonstrating the utility of semiconductor nanocrystals for physicalmapping positional cloning.

Example 6 RNA-Based FISH Assay

The specimen for FISH assay is prepared using various published methodsdepending on the specimen type, as described above.

Semiconductor nanocrystal-FISH probe(s) for RNA are denatured to theirsingle-stranded form. The specimen and probes in single-stranded formare allowed to hybridized to their complimentary sequence inhybridization buffer optionally containing blocking DNA (e.g., 5 minutesto overnight at 25° C.-70° C.).

The excess semiconductor nanocrystal-FISH probes are removed using aseries of stringent washes (e.g. 0.4×SSC to 2×SSC with or withoutformamide at 25° C.-72° C.). Optionally, the specimen are air dried andcounterstained to localize the nuclei.

The fluorescence signals of the semiconductor nanocrystal-FISH probesare viewed under an epi-fluorescence microscope. Alternatively, thesignals can be measured using a laser detector.

In particular, synthetic oligonucleotide probes directed against acertain mRNA are labeled with semiconductor nanocrystals at the 3′ or 5′end. The probes are added to the hybridization mix containing 50%formamide, 10% dextran sulphate, 1% sarkosyl, 0.02 M sodium phosphate,4×SSC, 1×Denhardt's solution and 10 mg/ml ssDNA. The probe mixture isadded to the specimen, a coverslip is placed over the probe, and theprobe and mRNA are allowed to hybridized for 16-20 hours at 42° C. in ahumidified chamber. After hybridization, the excess probes are removedby washing several times in 1×SSC at 55° C. and air-dried. The slidesare then examined under a fluorescence microscope.

Example 7 Multiplexed DNA- and RNA-Based FISH Assay

The specimen for FISH assay is prepared using various published methodsdepending on the specimen type, as described above.

Double-stranded specimen DNA and semiconductor nanocrystal-FISH probe(s)can be denatured together or separately to their single-stranded form.

The specimen and semiconductor nanocrystal-FISH probes for both RNA andDNA in single-stranded form are allowed to their complimentary sequencein hybridization buffer optionally containing blocking DNA (5 minutes toovernight at 25° C.-70° C.).

The excess semiconductor nanocrystal-FISH probes for RNA and DNA areremoved using a series of stringent washes (e.g., 0.4×SSC to 2×SSC withor without formamide at 37° C.-72° C.).

A flow cytometer or other similar detector can be used to scan thespecimens to detect the fluorescence from the semiconductor nanocrystalRNA probe and the cell's DNA fluorescence signals are detectedsimultaneously. Alternatively, the rare cells are sorted aside forfurther DNA analysis using a microscope.

For example, semiconductor nanocrystal-FISH probes for DNA and RNA areadded to the hybridization mix containing 50% formamide, 10% dextransulphate, 1-4×SSC (saline citrate), 3 μg Cot1 DNA, 10 mg/ml ssDNA. Theprobe mixture is deposited on the slide and a coverslip is placed on theprobe. The slide is placed on a hot plate for 3 minutes at 72° C. todenature the specimen and probe DNA. The specimen and probes insingle-stranded form are allowed to hybridize to their complimentarysequence overnight at 25° C. to 45° C. After hybridization, the excessprobes are removed by washing several times in 1×SSC at 55° C. andair-dried. The specimen is counterstained to localize the nuclei.

The fluorescence signals of the semiconductor nanocrystal-FISH probesare viewed under an epi-fluorescence microscope. Alternatively, thesignals are measured using a laser detector.

Example 8 Immunostaining and FISH Assays

Specimen cells are labeled with optimized concentration of semiconductornanocrystals-antibody(ies) or antibody with other dye label specific tocell surface protein of interest (e.g. anti-CD4 or anti CD14).

The cells are fixed and permeabilized by addition of reagents such asPERMEAFIX® (Ortho Diagnostics). After incubation, the cells are pelletedand washed.

The cell pellet is resuspended for FISH assay with hybridization buffercontaining single-strand semiconductor nanocrystal-RNA probes optionallycontaining blocking DNA. The mixture is allowed to hybridized to theircomplimentary sequence (5 minutes to overnight at 25° C.-70° C.).

The excess semiconductor nanocrystal-FISH probes are removed using aseries of stringent washes (e.g., 0.4×SSC to 2×SSC with or withoutformamide at 37° C.-72° C.).

A flow cytometer or other similar detector can be used to scan thespecimens to sort and/or to detect the surface immunofluorescence andfluorescence from the semiconductor nanocrystal RNA probesimultaneously.

In particular, specimen cells are fixed and permeabilized by addition ofreagents such as Penneafix (Ortho Diagnostics). After incubation thecells are pelleted and washed. The cell pellet is resuspended for FISHassay in hybridization buffer. The hybridization mixture containssemiconductor nanocrystal-FISH probes for DNA or RNA, 50% formamide, 10%dextran sulphate, 14×SSC, 3 μg Cot1 DNA (for DNA probes) and or 10 mg/mlssDNA(for RNA probes). The mixture is allowed to denatured for 2 minutesat 72° C. and hybridized overnight at 25° C.-45° C. The excess FISHprobes are removed by washing several times in 1×SSC at 55° C.Monoclonal antibody from mouse to a protein of interest is used to bindthe specimen cells using standard immunohistochemistry procedures. Theexcess antibody is removed, the specimen is incubated with semiconductornanocrystal-labeled anti-mouse IgG. The excess semiconductor nanocrystalanti-mouse IgG is removed and the specimen cells can be examined under afluorescence microscope. Alternatively, a flow cytometer or othersimilar detector can be used to scan the specimens to sort and/or todetect the fluorescence from the semiconductor nanocrystal-FISH probesand semiconductor nanocrystal-labeled antibody.

Example 9 FISH Assay of Amplified Specimen DNA

Specimen cells were fixed and permeabilized by addition of reagents suchas Permeafix (Ortho Diagnostics). After incubation the cells arepelleted and washed.

The cell pellet is resuspended with PCR reaction mixture along withoptimized concentrations of dNTP and semiconductor nanocrystal dUTP,semiconductor nanocrystal-forward primer and semiconductornanocrystal-reverse primer for DNA or RNA of interest, Taq polymeraseand gelatin.

The DNA in the above reaction mixture is amplified in a thermocyclerprogrammed for the specific conditions.

After in vitro amplification, cells are pelleted and resuspended inhybridization mix along with semiconductor nanocrystal-oligonucleotideprobe directed against the specific amplified product and, optionally,blocking DNA to reduce background noise.

The product DNA is denatured and allowed to hybridized with thesemiconductor nanocrystal-oligonucleotide probe (5 minutes to overnightat 25° C.-70° C.).

The excess semiconductor nanocrystal-oligonucleotide probes are removedusing a series of stringent washes (e.g., 0.4×SSC to 2×SSC with orwithout formamide at 37° C.-72° C.).

A flow cytometer, or other similar detector, or epi fluorescencemicroscope is used to detect the fluorescence signals from thesemiconductor nanocrystal-oligonucleotide probes.

Example 10 Signal Amplification in a DNA Hybridization Assay

A unique DNA sequence is chosen as a capture probe and is designed tohave a sequence complementary to a sequence in a capture extenderoligonucleotide. This sequence can range from 10 to 25 nucleotides inlength. This DNA capture probe sequence is chemically synthesized andpurified.

The capture probe is conjugated to the surface of a well of a microtiterplate, using any one of a variety of standard chemical linkages andmethods well known in the art. The analyte is immobilized on themicrotiter support through one or more oligonucleotides (captureextenders) that have first and second complementary sequences. The firstsequence is complementary to a sequence in the target analyte and thesecond sequence is complementary to a sequence in the support-boundcapture probe. The capture extenders and the sample can be added to theassay simultaneously. Alternatively, the capture extender can be addedand allowed to hybridize to the complementary sequence in thesupport-bound capture probe prior to addition of the sample.Hybridization is allowed to occur under conditions that favorhybridization for a period of time sufficient to effect hybridization ofthe capture extender to the capture probe and the target analyte to thecapture extender. The assay format can be designed so that the targetanalyte will be immobilized to the solid support only if two captureextenders, in which the first complementary sequences thereof arecomplementary to distinct segments in the target analyte, hybridize tothe target. In addition, the support-bound capture probe and the captureextenders can be designed so that the second complementary sequence inthe capture extenders are complementary to distinct segments in acapture probe. Thus, in order to immobilize the target to the solidsupport, the second complementary sequences of two distinct captureextenders hybridize to distinct complementary sequences in a captureprobe and the first complementary sequences of the capture extendershybridized to distinct sequences in the target analyte.

After hybridization, an optional step is performed under appropriatestringency conditions to separate material not retained on the solidsupport.

One or more label extender oligonucleotide probes complementary to theanalyte sequence is chosen. Each of these target probe oligonucleotidescontains a second sequence segment substantially complementary to anucleic acid sequence within an amplifier oligo. Hybridization isallowed to occur. After hybridization, a wash step is performed underappropriate stringency conditions.

The amplification oligo contains a sequence complementary to a portionof each target probe oligo and also contains a multiplicity of labelprobe sites. Hybridization is allowed to occur. After hybridization, awash step is performed under appropriate stringency conditions.

Semiconductor nanocrystal-conjugated oligos which are complementary tothe amplifier oligos are allowed to hybridize to the amplifier oligos.Hybridization is allowed to occur. After hybridization, a wash step isperformed under appropriate stringency conditions.

Finally the well is scanned in a fluorimeter. The amount of lightemitted at the excitation wavelength of the semiconductor nanocrystalsis measured. Light emission at the detection wavelength is proportionalto the amount of target nucleic acid present.

Example 11 Signal Amplification in a Biotin/Avidin-Layered AmplificationAssay

The sample is immobilized on a solid support by established techniques.Unbound sample is washed from the solid support.

A biotin-labeled antibody is reacted with an immobilized antigen,followed by appropriate washes. Biotin-conjugated semiconductornanocrystals are mixed with avidin (or streptavidin) in specific ratiosto form complexes consisting of multiple inter-linkedavidin-semiconductor nanocrystal complexes with some free biotin-bindingsites still available. An aliquot of this complex is added to theimmobilized biotin-labeled antibody/antigen complex. Unbound complex iswashed using appropriate conditions.

Result for assay is determined by measuring the intensity of lightemitted at wavelengths that correspond to the semiconductor nanocrystalsused.

Example 12 Multiplexed Bead-Based Assay Using Multiple SemiconductorNanocrystal-Ligand Receptor Pairs

5×10⁶ 10 μm polystyrene-divinylbenzene, diaminododecane beads are rinsedwith ethanol, then twice with phosphate buffered saline (PBS) with 0.05%NaN₃. They are then resuspended in 1 ml of this solution, and incubatedfor one hour sequentially with each of the following reagents, with twowash steps between each incubation: 50 μg/ml BSA-Biotin (Bovine serumalbumin-biotinylated) in PBS/NaN₃, 50 μg/ml streptavidin in PBS/NaN3, 50μg/ml of a biotinylated monoclonal antibody which specifically binds thenoninteracting region of the receptors (e.g., MAb179), and 2 μg/ml ofthe purified extracellular domain of the receptor (e.g., IL-5).

Another 5×10⁶ 10 μm polystyrene-divinylbenzene, diaminododecane beadsare rinsed with ethanol, then twice with PBS with 0.05% NaN₃. They arethen resuspended in 1 ml of this solution, and incubated for one hoursequentially with each of the following reagents, with two wash stepsbetween each incubation: 50 μg/ml BSA-Biotin in PBS/NaN₃, 50 μg/mlstreptavidin in PBS/NaN₃, 50 μg/ml of a biotinylated monoclonal antibodywhich specifically binds the noninteracting region of the receptors(e.g., MAb179), and 2 μg/ml of the purified extracellular domain of thereceptor (e.g., IL-1).

2×10⁶ of each receptor bound bead are thoroughly mixed into a singletube. This suspension was distributed into 8 wells containing 5000 beadseach. 5000 beads of each of the receptors (IL-1 and IL-5) are put intotwo further sets of 8 wells, as shown in FIG. 3.

IL-1 receptor-antagonist and IL-5 receptor antagonist are conjugated tored and green semiconductor nanocrystals using techniques previouslydescribed. These are prepared in a stock solution to 16 nM, then mixedin equal volumes with a dilution series (0, 1 nM, 10 nM, 100 nM, 1 μM,10 μM, 100 μM, 1 mM) of a competing molecule. 50 μl of these solutionsare added to each of the wells, each well 1-8 containing a uniqueconcentration of the inhibitor (as above), each of the columns A-C (FIG.3) having identical conditions and only different beads. Afterincubation, the beads from each well are transferred to tubes for flowcytometric analysis, though other analysis techniques may be preferable.The cytometer separates the beads into individual events, allowing thedetection of two wavelengths of light (e.g. the fluorescence from thered and green semiconductor nanocrystals) simultaneously upon excitationwith a 488 nm laser. In this assay, the ligand can inhibit one, both, orneither of the ligand-receptor pairs. If the inhibitor is ineffectivefor both systems, the results will be uniform across the concentrationof inhibitor, and should vary very little from the control wells. Whenthe inhibitor is effective in only one of these assays, for instanceagainst IL-1 only, then the wells in column A have decreasing redfluorescence associated with the beads at higher inhibitorconcentrations.

A plot of the relative fluorescence with respect to the standard as afunction of concentration reveals the concentration at which the normalreceptor-ligand pair binding is inhibited by 50% (this is known as theIC50). In contrast, the wells in column C (FIG. 3) are unaffected by thepresence of the inhibitor at all concentrations. In the center lane, thetrue multiplexed assay is performed, and the results are seen as beadswhich fluoresce lower intensities of red at the higher concentrations(due to inhibition of binding), and beads which fluoresce with ahomogeneous intensity of green (due to the lack of inhibition of theIL-5 interactions). Homogeneously stained green beads are presentthroughout each of the inhibition series of the red emitting beads.

This format has advantages over standard formats using dye molecules inthat the number may be expanded significantly beyond two using a varietyof different colors (and thus different size or composition)semiconductor nanocrystals, each conjugated to a unique receptor orligand, in order to maximize the throughput in a single well.Furthermore, the fact that each and every scintillation event is due toa ligand-receptor pair formation (in the low concentration limit) makesthis a very sensitive technique for which there are no standardmultiplexing methods.

Example 13 Use of Semiconductor Nanocrystals in a HomogeneousScintillation Inhibition Assay

When the semiconductor nanocrystals are brought within 1 μm of aradioactive nucleus, they can be made to emit light upon the decay ofthat nucleus, which produces high energy radiation, which is dissipatedby the solvent, and can excite the semiconductor nanocrystals, causingthem to emit light. In order to get the semiconductor nanocrystals toefficiently emit light, it is necessary to have the nucleus remain inclose proximity. This can be accomplished through ligand-receptorinteractions, where one is radiolabeled, and the other is conjugated toa semiconductor nanocrystal. This homogeneous assay format isadvantageous in that it allows free association in solution of all thereceptor-ligand pairs, each of which can be prepared to scintillate in aunique region of the spectrum. Semiconductor nanocrystals which emit ingreen, yellow, and red regions of the spectrum, and are well resolvedspectrally are conjugated to three different ligands (receptorantagonists) to the IL-1, IL-5, and IL-12 receptors respectively. Asolution is prepared which is 1 nM in each of these semiconductornanocrystal-receptor antagonist conjugates. Aliquots are mixed with aconcentration series of the inhibitor of interest, and brought toidentical final volumes and concentrations of RA-semiconductornanocrystals (0.75 nM each). Radiolabeled (³H) IL-1, IL-5, and IL-12receptor extracellular domains are prepared by proton exchange, as knownin the art. A stock is prepared which contains 1 nM of all three ofthese tritiated proteins in phosphate buffered saline. This stock isadded to result in a final concentration of each receptor of 0.001 nM, alarge excess of ligand over receptor. Scintillation is seen only fromsemiconductor nanocrystals which are bound to receptor antagonist whichis not inhibited by the candidate inhibitor. A lower signal is seen inthe characteristic color when the inhibition is effective. These may bereferenced to the control well without inhibitor.

Signal may be detected in a dark box with a camera mounted such that itcan image the entire plate. All that is necessary is for the camera tointegrate the collected light for a long period, as the scintillation isa low background technique. In addition, multiple filters can be used toseparate the signal arising from each ligand-receptor pair. In FIG. 4,lanes A, B, and C contain different inhibitors, lane A for IL-1 only,lane B for IL-5 and IL-12, and lane C for all receptors.

The unique combination of colors which indicate inhibition willdetermine which receptors the candidate may be an appropriate drug for.One principal advantage of such a system is that it may be performedwith as many ligands as one can produce spectrally resolvedsemiconductor nanocrystals. Since the range of 300 nm to 1.5 μm isavailable for semiconductor nanocrystal manufacture, well over 20 colorsshould be available for multiplexing in a homogeneous reaction format.Furthermore, the fact that each and every scintillation event is due toa ligand-receptor pair formation (in the low concentration limit), thisis a very sensitive technique for which there are now no knowntechniques that are capable of multiplexing.

Example 14 Hybridization Assays

Hybridization assays are a convenient method for determining thepresence or absence of a specific DNA sequence in a sample.Semiconductor nanocrystals are used to 5′ end label a pair of allelespecific oligonucleotide primers. These primers are used in PCR or otherDNA amplification reaction, such that the target sequence of interest isamplified, i.e., increased, in number. A sequence-specificoligonucleotide probe is synthesized, and is immobilized onto a surfaceto become a capture probe. The amplified target is applied to thesurface, and allowed to hybridize under sufficiently stringentconditions of temperature and salt concentration. After hybridization,unbound material is washed away. The presence of semiconductornanocrystals immobilized on the surface is detected by exciting at anallowed absorbance wavelength, and measuring at the semiconductornanocrystal emission wavelength. The presence of a semiconductornanocrystal emission on the surface is directly proportional to theamount of amplified target DNA present.

A. A DNA sequence complementary to the target sequence is chosen. Thissequence can range from 15 to 25 nucleotides in length. This DNA probesequence is chemically synthesized and purified. The probe is conjugatedto the surface of a well of a microtiter plate, using any one of avariety of standard chemical linkages.

Two PCR primer sequences are selected, such that they specificallyamplify a portion of the target DNA sequence containing the region to beprobed. The 5′ end of each of the two primers is labeled with asemiconductor nanocrystal with peak emission at 525 nm. These twoprimers are used to amplify a defined portion of the target DNA in astandard PCR reaction. Since the semiconductor nanocrystal is conjugatedto the primer, and the primer is extended to become the amplifiedtarget, the semiconductor nanocrystal is connected to the 5′ end of theamplified target.

After the thermal cycling the target can be denatured or madesingle-stranded by heating it for two minutes at 95° C.

The hybridization is performed by adding 20 μl of the amplified targetand 480 μl of 5×SSPE Buffer to the microtiter well coated with DNAprobe, and incubating for 60 minutes at 37° C. After hybridization, awash step under stringent conditions is performed by adding 500 μl of3×SSPE, and incubating for 15 minutes at 37° C. The well is rinsed twicewith water.

In places where the amplified DNA hybridized to the immobilized probe,the semiconductor nanocrystal with 525 emission is connected to thesurface. The well is scanned in a fluorimeter. UV light is shone uponthe well, and the amount of phosphorescence at 525 nm is measured. Thepresence of 525 nm emission indicates that the immobilizedprobe-captured semiconductor nanocrystal-labeled amplified target, andremained hybridized after the stringent wash and water rinse steps.

B. Three DNA sequences complementary to the three different targetsequences are chosen. These sequences can range from 15 to 25nucleotides in length. These three DNA probe sequences are chemicallysynthesized and purified.

These three probe sequences are blended, and are conjugated to thesurface of the well of a microtiter plate, using any one of a variety ofstandard chemical linkages.

Two PCR primer sequences are selected for each of three targets. Theseprimers are chosen such that each pair will specifically amplify aportion of the target DNA sequence containing the region complementaryto the three DNA probes. The 5′ end of each of the first pair of primersis labeled with a semiconductor nanocrystal with peak emission at 525nm. The 5′ end of each of the second pair of primers is labeled with asemiconductor nanocrystal with peak emission at 580 nm. The 5′ end ofeach of the third pair of primers is labeled with a semiconductornanocrystal with peak emission at 650 nm.

These three primer pairs are blended, and are used to simultaneouslyamplify three defined portions of the target DNA in a standard PCRreaction. Since each of the three semiconductor nanocrystal types areconjugated to their respective primers, and the primers are extended tobecome the amplified target, each of the three amplified targetsequences are labeled with the semiconductor nanocrystal color withwhich their respective primers were labeled.

After the thermal cycling the amplified target can be denatured or madesingle-stranded by heating for two minutes at 95° C.

The hybridization is performed by adding 20 μl of the amplified targetand 480 μl of 5×SSPE Buffer to the microtiter well coated with threedifferent sequences of DNA probe, and incubating for 60 minutes at 37°C. After hybridization, a wash step under stringent conditions isperformed by adding 500 μl of 3×SSPE, and incubating for 15 minutes at37° C. The well is rinsed twice with water.

The well is scanned in a fluorimeter. UV light is shone upon the well,and the amount of phosphorescence at 525, 580 and 650 nm wavelengths ismeasured. The presence of 525 nm emission indicates that the immobilizedprobe captured the first semiconductor nanocrystal labeled amplifiedtarget, and remained hybridized after the stringent wash and water rinsesteps. The presence of 580 nm emission indicates that the immobilizedprobe captured the second semiconductor nanocrystal-labeled amplifiedtarget. The presence of 650 nm emission indicates that the immobilizedprobe captured the third semiconductor nanocrystal-labeled amplifiedtarget.

This multiplex assay allows simultaneous amplification, labeling anddetection of three different targets.

Example 15 Sequence-Specific Extension

Sequence-specific extension assays are a convenient method fordetermining the presence or absence of a specific DNA sequence in asample. Two PCR primers are chosen, and the region of interest such asthe site of a Single Nucleotide Polymorphism (SNP) is PCR-amplified. Asequence-specific extension primer is chosen, such that the 3′ end ofthe primer either ends immediately over the site of the SNP, or iswithin 5 nucleotides of the SNP. This primer is conjugated via its 5′end to a surface, such as a microtiter plate well or bead. In theabsence of complete sequence complementarity, the base mismatch willdisrupt the double-stranded complex formed by the sequence-specificextension primer and the PCR amplified target. The DNA polymerase has nosite for attachment, so extension will not occur. If the primer andamplified target sequences are complementary, sequence-specificextension will occur. If the extension reaction mixture includes alabeled dNTP (e.g., semiconductor nanocrystal-labeled dGTP), thepresence or absence of extension can be detected. This allows adetermination of the original target sequence.

Two different DNA sequence specific primer sequences are chosen, suchthat the 3′ end of the primers overlaps a SNP in the target DNA sequenceby 2 nucleotides. These sequence can range from 15 to 25 nucleotides inlength. The first sequence will be completely complementary to allele A,and will be called primer A. The second sequence will be completelycomplementary to allele B, and will be called primer B.

Primer A and Primer B sequences are chemically synthesized and purified.The 5′ end of primer A is conjugated to the surface of the well of amicrotiter plate, using any one of a variety of standard chemicallinkages.

The 5′ end of this primer B is conjugated to the surface of a secondwell of a microtiter plate, using any one of a variety of standardchemical linkages. A semiconductor nanocrystal-dGTP conjugate isprepared and purified. The semiconductor nanocrystal is chosen with 525nm emission. Two PCR primer sequences are selected, such that theyspecifically amplify a portion of the target DNA sequence containing theSNP to be evaluated. These two primers are used to amplify a definedportion of the target DNA in a standard PCR reaction (AmpliTaqPolymerase, dNTPs, Magnesium Chloride, buffer and target DNA).

The sequence-specific extension is performed by combining 2.5 U AmpliTaqDNA polymerase, 2.5 mM magnesium chloride, buffer, 200 μM dATP, 200 μMdTTP, 200 μM dCTP, 20 μM dGTP, and 200 μM semiconductor nanocrystal-dGTPconjugate. 95 μl of this solution is added to the microtiter well coatedwith sequence-specific primer.

After the thermal cycling, the target is denatured or madesingle-stranded by heating for two minutes at 95° C. Add 5 μl ofdenatured target to the 95 μl of reaction solution contained in thewells of the microtiter plate. The extension reaction is allowed tooccur for 5 minutes at 60° C.

After extension, a wash step under denaturing conditions is performed byadding 500 μl of 3×SSPE, and incubating for 5 minutes at 95° C. Rinsethe well twice with water.

In places where the amplified DNA target annealed to the immobilizedprimer and extended, the semiconductor nanocrystal with 525 emission isincorporated into the extension sequence, and is thus connected to thesurface of the plate via the sequence-specific hybridization. The wellis scanned in a fluorimeter. UV light is shone upon the well, and theamount of phosphorescence at 525 nm is measured.

The presence of 525 nm emission indicates that the immobilized probecaptured semiconductor nanocrystal-labeled amplified target, andremained hybridized after the stringent wash and water rinse steps.

Example 16 Oligonucleotide Ligation Assay

Two oligonucleotide probes are hybridized to adjacent sequences of PCRamplified target DNA with the join situated at the position of a knownmutation site. DNA ligase covalently joins the two oligonucleotideprobes only if they are perfectly hybridized, which will occur if nomutation is present. Initial studies tagged one probe with biotin andthe other with a reporter molecule such as digoxygenin. On transfer tostreptavidin-coated microtiter plates washing will remove the reporterlabeled probe unless ligation has occurred. Signal detection willtherefore only occur if the mutation is not present. This technology wasimproved by a method of typing two alleles in a single microtiter well,by marking each of the allele-specific primers with different enzymereporter molecules so that two different colors can be produced.Advances to the ligation technique have replaced the biotin withmobility modifying tails and digoxygenin with fluorescent tags (e.g.,FAM, HEX or TET) with the ligation products being electrophoresed andanalyzed on an automated sequencer. These modifications have allowed thenumber of known point mutation sites which can be screened at any onetime to be increased.

An oligonucleotide sequence complementary to the wild type of the targetsequence is chosen. The 3′ end of this oligo ends with the lastnucleotide over a Single Nucleotide Polymorphism (SNP). This sequencecan range from 15 to 25 nucleotides in length.

A second oligonucleotide sequence is chosen, which is complementary tothe mutant sequence of the same SNP. The 3′ end of this oligo also endswith the last nucleotide over the SNP site. This sequence can range from15 to 25 nucleotides in length.

These two DNA probe sequences are chemically synthesized and purified.These oligo probes are separately conjugated by their 5′ end to thesurface of the wells of a microtiter plate, using any one of a varietyof standard chemical linkages.

A single ligation probe sequence is selected. The 5′ end of this probewill be directly adjacent to the 3′ end of the two selectedoligonucleotide sequences. This sequence can range from 10 to 25nucleotides in length.

The ligation probe is chemically synthesized, and the 5′ end isphosphorylated. The 3′ end of the ligation probe is conjugated to asemiconductor nanocrystal with emission at 525 nm.

A ligase reaction is performed in each of the two wells containing thetwo sequences of oligonucleotides. This reaction consists of theligation probe, denatured or single-stranded target DNA, T7 ligase,magnesium chloride and buffer in an aqueous solution.

The plate can be thermally cycled 25 times from 60° C. to 95° C., with adwell time of 1 minute at each temperature to achieve arithmeticamplification of the target.

In the presence of the complementary target sequence, the target acts asa template for the ligase reaction. The sequence-specificoligonucleotide and the ligation probe both anneal to thesingle-stranded target DNA. The T7 enzyme ligates the 3′ end of thesequence-specific oligo to the phosphorylated 5′ end of the ligationprobe.

In the case where the template DNA is not complementary to thesequence-specific oligonucleotide, the mismatch at the 3′ end of theannealed specific oligo is sufficient to prevent the ligation reaction.

The wells of the microtiter plate are washed 3 times with water.

In wells where the DNA ligation reaction occurred, the semiconductornanocrystal with 525 nm emission will be immobilized to the bottom ofthe well. The well is scanned in a fluorimeter. UV light is shone uponthe well, and the amount of phosphorescence at 525 nm is measured.

The presence of 525 nm emission indicates that the ligationprobe/semiconductor nanocrystal-conjugate was immobilized to the surfaceof the well via the ligation reaction, and remained after the waterrinse steps.

Example 17 Tissue Microarrays for High-Throughput ImmunohistochemicalStaining of Tumor Specimens

Tissue microarrays were prepared as described in Kononen et al. (1998)Nat. Med. 4:844-847. The tumor microarray block was cut into 3-8 μmsections, deparaffinized and prepared for immunochemistry by usingconventional techniques. Monoclonal antibody from mouse to cytokeratin8/18 was used to bind the cytokeratin inside the cells. The excessantibody was removed, the specimen was incubated with biotinylatedanti-mouse IgG followed by 40 nM semiconductor nanocrystal-streptavidin.The excess semiconductor nanocrystal-streptavidin was removed and theslides examined under a fluorescence microscope.

The staining pattern of the cells was readily visualized using afluorescence microscope.

Example 18 Use of Semiconductor Nanocrystals in Histochemical andCytochemical Analyses

As explained above, semiconductor nanocrystals may be used to domultiple analysis staining on a tissue sample or blood sample or anysample requiring multiplexed analysis of cellular or extracellularmarkers. The procedure is carried out in a two-step reaction whereby aprimary antibody is followed by a semiconductor nanocrystal-conjugatedantibody or by using an antibody (or other biomolecule) semi-conductornanocrystal conjugate to directly label the sample. For example five ormore different populations of semiconductor nanocrystals are synthesizedwith emission spectra that spaced at 40 nm intervals from 490-650 nm.Each spectrally distinct population of semiconductor nanocrystal isconjugated to a different molecule which specifically recognizes thebiomolecule of interest which may or may not be present in the sample tobe analyzed. Following standard staining protocols, the sample islabeled with the semiconductor nanocrystals and analyzed for thelocation and quantity of the target molecule.

A. The sample is prepared for immunohistochemical analysis. Theincubation buffer is PBS+0.1% BSA+NaN₃ (or similar buffer). Activegroups are blocked. The sample is washed and incubated withsemiconductor nanocrystal conjugates (the number dependent on the numberof parameters being assessed). The sample is then washed, mounted andanalyzed using conventional fluorescent microscopy techniques or aspectral scanning device as described above.

B. Cellular compounds that are known to exist in spatially distinctregions of a cell are labeled with the same six colors. In this case,six different compounds can be unambiguously monitored in each distinctlocation within the cell.

C. Compounds in spatially distinct regions of the cell are labeled withdifferent sets of 6 colors, where each set has a small wavelength shiftrelative to the others. For instance, proteins located in the nucleusare labeled with semiconductor nanocrystals that emit at 500, 530, 560,590, 620 and 650 nm, while proteins located in the cellular membrane arelabeled with 505, 535, 565, 595, 625 and 655 nm emitting semiconductornanocrystals. The advantage to using nonidentical sets of colors is thatif the location of compounds is not known, this type of “barcoding” ofthe different cellular compounds can be used to unambiguously determinethe location of each. A problem arises only when nonspectrally distinctsemiconductor nanocrystals (e.g. 550 and 555) colocalize. If thishappens, however, the information can be used in a second experimentwhere the labels on the colocalized compounds are rearranged toeliminate the spectral overlap. For instance, in the example ofcolocalized 550 and 555 nm emitting semiconductor nanocrystals, the 555nm label is replaced in a second experiment with 590 nm emittingsemiconductor nanocrystals. By barcoding different cellular compounds,it may be possible with CdSe semiconductor nanocrystals to track thelocation of as many as 150 distinct compounds within a cell. Using othersemi-conductor nanocrystal materials such as CdTe, InP, InAs, CdS, etc.can dramatically increase this number.

Example 19 Competitive Microsphere Filter Assay

An explained above, semiconductor nanocrystals can be used incompetitive microsphere filter assays, such as competitive lateximmunoassays. In this application the detection agent is a semiconductornanocrystal or a semiconductor nanocrystal-encoded solid conjugate. Theconjugate is a molecule that specifically recognizes the analyte. Theantigen conjugates are direct semiconductor nanocrystal-antigenconjugates or semiconductor nanocrystal-dyed microsphere-conjugates thatare small enough to pass through filter pores.

This allows multiple simultaneous detections using a light source forexcitation of the semiconductor nanocrystals and detection of emissions.The detection takes place on the filter or in the filtrate and the assaymay be carried out in a high throughput multiwell environment. Thefilters in this format are opaque to the excitation light and allowdetection of semiconductor nanocrystals in the filtrate without the needfor washes or sample removal.

In particular, antibodies to a specific analyte are immobilized onmicrospheres with a diameter greater than that of the filter pores usingstandard adsorption or conjugation technologies. The antibody conjugatesare placed in an appropriate buffer (e.g. PBS) in the upper chamber orinsert in a 96-well plate separated from the bottom of the plate by alight impermeant porous filter (e.g. BD fluoblock filters). The mediumcontaining the analyte(s) and a standard amount of analyte(s) labeledwith semiconductor nanocrystals that have a diameter less than that ofthe filter pores are added to the upper chamber or insert. After anappropriate incubation time (e.g., 30 seconds to 24 hours) the amount offluorescence in the lower chamber is quantitated by spectral analysissystems described above.

This assay provides information on the concentration of analyte(s) inany biological medium. It is especially useful for measuring multipleanalytes in a diagnostic type test and may also be used forenvironmental, research or screening applications. The greater theconcentration of analyte in the sample the greater the amount offluorescence detected in the lower chamber. Thus, this system provides adesirable increasing curve in terms of analyte concentration.

FIG. 5 is a pictorial representation of a competitive microsphere filterassay as described herein.

Thus, novel methods for using semiconductor nanocrystals are disclosed.Although preferred embodiments of the subject invention have beendescribed in some detail, it is understood that obvious variations canbe made without departing from the spirit and the scope of the inventionas defined by the appended claims.

1. A method of detecting one or more target analytes in a samplecontaining or suspected of containing the one or more analytes,comprising the steps of: (a) providing the sample on a solid support;(b) combining said sample with a semiconductor nanocrystal conjugate,wherein said combining is performed under conditions that allowformation of a complex comprising said conjugate and said analyte, whenpresent; (c) removing any unbound conjugate; (d) detecting the presenceof the complex, if present, by monitoring a spectral emission mediatedby the semiconductor nanocrystal in the complex, wherein the emissionindicates the presence of one or more target analytes in the sample. 2.The method of claim 1, wherein there is a plurality of target analytesand the method further comprises: (a) providing a conjugate specific foreach target analyte, wherein each semiconductor nanocrystal conjugatehas an emission spectrum distinct from the other semiconductornanocrystal conjugates; and (b) detecting the presence of the targetanalytes by monitoring the spectral emissions of the sample, wherein theemissions indicate the presence of the target analytes in the sample. 3.The method of claim 1, wherein the one or more analytes is a nucleicacid molecule.
 4. The method of claim 3, wherein the nucleic acidmolecule is contained within a chromosome or chromosomal fragment. 5.The method of claim 2, wherein the one or more analytes is a nucleicacid molecule.
 6. The method of claim 5, wherein the nucleic acidmolecule is contained within a chromosome or chromosomal fragment. 7.The method of claim 6, wherein the target analytes are present on morethan one chromosome or chromosomal fragments.
 8. The method of claim 7,wherein each of the target analytes is different from the others.
 9. Themethod of claim 6, wherein the target analytes are present on a singlechromosome.
 10. The method of claim 9, wherein each of the targetanalytes is different from the others.
 11. The method of claim 3,wherein the nucleic acid molecule is a DNA molecule.
 12. The method ofclaim 3, wherein the nucleic acid molecule is a RNA molecule.
 13. Themethod of claim 3, wherein the conjugate comprises at least onepolymerase chain reaction primer.
 14. The method of claim 1, wherein theone or more analytes is a polypeptide.
 15. The method of claim 14,wherein the semiconductor nanocrystal conjugate comprises an antibody.16. The method of claim 1, wherein the semiconductor nanocrystalconjugate comprises an aptamer.
 17. A method of detecting one or moretarget analytes in a sample containing or suspected of containing theone or more analytes, comprising the steps of: (a) providing anunlabeled specific-binding molecule on a solid support; (b) combiningsaid sample with said specific-binding molecule, wherein said combiningis performed under conditions that allow formation of a first complexcomprising said specific-binding molecule and said analyte, whenpresent; (c) removing any unbound sample; (d) combining said firstcomplex with a semiconductor nanocrystal conjugate, wherein saidcombining is performed under conditions that allow formation of a secondcomplex comprising said conjugate and said analyte, when present; (e)removing any unbound conjugate; (f) detecting the presence of the secondcomplex, if present, by monitoring a spectral emission mediated by thesemiconductor nanocrystal in the second complex, wherein the emissionindicates the presence of one or more target analytes in the sample. 18.The method of claim 17, wherein there is a plurality of target analytesand the method further comprises: (a) providing an unlabeledspecific-binding molecule on a solid support specific for each targetanalyte and providing a semiconductor nanocrystal conjugate specific foreach target analyte, wherein each semiconductor nanocrystal conjugatehas an emission spectrum distinct from the other semiconductornanocrystal conjugates; and (b) detecting the presence of the targetanalytes by monitoring the spectral emissions of the sample, wherein theemissions indicate the presence of the target analytes in the sample.19. The method of claim 17, wherein the one or more analytes is anucleic acid molecule.
 20. The method of claim 19, wherein the nucleicacid molecule is a DNA molecule.
 21. The method of claim 19, wherein thenucleic acid molecule is a RNA molecule.
 22. The method of claim 17,wherein the one or more analytes is a polypeptide.
 23. The method ofclaim 17, wherein the semiconductor nanocrystal conjugate comprises anantibody.
 24. The method of claim 17, wherein the semiconductornanocrystal conjugate comprises an aptamer.
 25. A method of detectingone or more target analytes in a sample containing or suspected ofcontaining the one or more analytes, comprising the steps of: (a)providing the sample on a solid support; (b) combining with said samplea specific-binding molecule, wherein (i) said specific-binding moleculecomprises a first member of a binding pair, and (ii) said combining isperformed under conditions that allow formation of a first complexcomprising said specific-binding molecule and said analyte, whenpresent; (c) removing any unbound specific-binding molecule; (d)combining said first complex with a second member of the binding pair,wherein (i) said second member of the binding pair is linked to a firstsemiconductor nanocrystal; and (ii) said combining is performed underconditions that allow formation of a second complex comprising thebinding pair and said one or more analytes; and (e) detecting thepresence of the second complex, if present, by monitoring a spectralemission mediated by the first semiconductor nanocrystal in the secondcomplex, wherein the emission indicates the presence of one or moretarget analytes in the sample.
 26. The method of claim 25, wherein theone or more analytes is a nucleic acid molecule.
 27. The method of claim26, wherein the nucleic acid molecule is contained within a chromosomeor chromosomal fragment.
 28. The method of claim 26, wherein the nucleicacid molecule is a DNA molecule.
 29. The method of claim 26, wherein thenucleic acid molecule is a RNA molecule.
 30. The method of claim 25,wherein the one or more analytes is a polynucleotide.
 31. The method ofclaim 30, wherein the specific-binding molecule is a polymerase chainreaction amplification product and said first member of the binding pairis incorporated in the amplification product.
 32. The method of claim26, wherein the specific-binding molecule is an aptamer.
 33. The methodof claim 25, wherein the one or more analytes is a polypeptide.
 34. Themethod of claim 33, wherein the first member of the binding pair is afirst antibody and the second member of the binding pair is a secondantibody reactive with the first antibody.
 35. The method of claim 25,wherein the first member of the binding pair is biotin and the secondmember of the binding pair is streptavidin.
 36. The method of claim 33,wherein the first member of the binding pair is biotin and the secondmember of the binding pair is streptavidin.
 37. The method of claim 25,wherein the first member of the binding pair is digoxygenin and thesecond member of the binding pair is an antibody directed againstdigoxygenin.
 38. The method of claim 25, wherein the first member of thebinding pair is flourescein and the second member of the binding pair isan antibody directed against flourescein.
 39. The method of claim 25,wherein there is more than one analyte, wherein the method furthercomprises: combining with said sample a second specific-bindingmolecule, wherein (i) said second specific-binding molecule comprises afirst member of a second binding pair, and (ii) said combining isperformed under conditions that allow formation of a third complexcomprising said second specific-binding molecule and said analyte, whenpresent; removing any unbound second specific-binding molecule;combining said third complex with a second member of the second bindingpair, wherein (i) said second member of the second binding pair islinked to a second semiconductor nanocrystal that has an emissionspectrum distinct from the first semiconductor nanocrystal; and (ii)said combining is performed under conditions that allow formation of afourth complex comprising the second binding pair and an analyte; anddetecting the presence of the fourth complex, if present, by monitoringa second spectral emission mediated by the second semiconductornanocrystal in the fourth complex, wherein the second emission indicatesthe presence of more than one target analyte in the sample.
 40. A methodof detecting one or more target analytes in a sample containing orsuspected of containing the one or more analytes, comprising the stepsof: (a) providing a first complex comprising at least onespecific-binding molecule to which is bound a semiconductor nanocrystalconjugate, wherein said semiconductor nanocrystal has a characteristicspectral emission and wherein said conjugate specifically binds to saidspecific-binding molecule; (b) combining said sample with said firstcomplex, wherein said combining is performed under conditions that allowformation of a second complex comprising said specific-binding moleculeand said analyte, when present; (c) detecting the presence of the secondcomplex, if present, by monitoring the characteristic spectral emissionof the semiconductor nanocrystal, wherein a change in the characteristicspectral emission indicates the presence of one or more target analytesin the sample.
 41. The method of claim 40, wherein a plurality of firstcomplexes are provided each comprising a different specific-bindingmolecule each bound to a conjugate which specifically binds eachspecific-binding molecule and where each specific-binding molecule bindsa different analyte, and wherein each conjugate bound to a differentspecific-binding molecule comprises a semiconductor nanocrystal that hasa characteristic spectral emission distinct from the other semiconductornanocrystals; and wherein changes in the spectral emission of anyselected semiconductor nanocrystal associated with a particularspecific-binding molecule in a first complex, indicates the presence ofan analyte that binds to the particular specific-binding molecule. 42.The method of claim 40, wherein said specific-binding molecule isradiolabeled, wherein when said conjugate is bound to saidspecific-binding molecule the semiconductor nanocrystal emits light. 43.The method of claim 42, wherein a plurality of first complexes areprovided each comprising a different specific-binding molecule eachbound to a conjugate which specifically binds each specific-bindingmolecule and where each specific-binding molecule binds a differentanalyte, and wherein each conjugate bound to a differentspecific-binding molecule comprises a semiconductor nanocrystal that hasa characteristic spectral emission distinct from the other semiconductornanocrystals; and wherein changes in the spectral emission of anyselected semiconductor nanocrystal associated with a particularspecific-binding molecule in a first complex, indicates the presence ofan analyte that binds to the particular specific-binding molecule. 44.The method of claim 40, wherein said specific-binding molecules areselected from the group consisting of a protein, an oligonucleotide, apolysaccharide or a small molecule.
 45. The method of claim 44, whereinsaid specific-binding molecules are receptor molecules and a conjugateis provided that binds to each receptor.